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• Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration. Brosch, Philippa K.; Korsa, Tessa; Taban, Danush; Eiring, Patrick; Hildebrand, Sascha; Neubauer, Julia; Zimmermann, Heiko; Sauer, Markus; Shirakashi, Ryo; Djuzenova, Cholpon S.; Sisario, Dmitri; Sukhorukov, Vladimir L. In Cancers, 14, S. 5794. 2022.
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  (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

  @article{brosch2022glucose, abstract = {(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.}, author = {Brosch, Philippa K. and Korsa, Tessa and Taban, Danush and Eiring, Patrick and Hildebrand, Sascha and Neubauer, Julia and Zimmermann, Heiko and Sauer, Markus and Shirakashi, Ryo and Djuzenova, Cholpon S. and Sisario, Dmitri and Sukhorukov, Vladimir L.}, journal = {Cancers}, keywords = {sauer}, pages = 5794, series = 23, title = {Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration}, volume = 14, year = 2022 }

  %0 Journal Article %1 brosch2022glucose %A Brosch, Philippa K. %A Korsa, Tessa %A Taban, Danush %A Eiring, Patrick %A Hildebrand, Sascha %A Neubauer, Julia %A Zimmermann, Heiko %A Sauer, Markus %A Shirakashi, Ryo %A Djuzenova, Cholpon S. %A Sisario, Dmitri %A Sukhorukov, Vladimir L. %B 23 %D 2022 %J Cancers %P 5794 %T Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration %U https://www.mdpi.com/2072-6694/14/23/5794 %V 14 %X (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

• Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis. Fink, Julian; Schumacher, Fabian; Schlegel, Jan; Stenzel, Philipp; Wigger, Dominik; Sauer, Markus; Kleuser, Burkhard; Seibel, Jürgen. In Org. Biomol. Chem., 19(10), S. 2203–2212. The Royal Society of Chemistry, 2021.
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  Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

  @article{fink2021azidosphinganine, abstract = {Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.}, author = {Fink, Julian and Schumacher, Fabian and Schlegel, Jan and Stenzel, Philipp and Wigger, Dominik and Sauer, Markus and Kleuser, Burkhard and Seibel, Jürgen}, journal = {Org. Biomol. Chem.}, keywords = {sauer}, number = 10, pages = {2203–2212}, publisher = {The Royal Society of Chemistry}, title = {Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis}, volume = 19, year = 2021 }

  %0 Journal Article %1 fink2021azidosphinganine %A Fink, Julian %A Schumacher, Fabian %A Schlegel, Jan %A Stenzel, Philipp %A Wigger, Dominik %A Sauer, Markus %A Kleuser, Burkhard %A Seibel, Jürgen %D 2021 %I The Royal Society of Chemistry %J Org. Biomol. Chem. %N 10 %P 2203--2212 %R 10.1039/D0OB02592E %T Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis %U http://dx.doi.org/10.1039/D0OB02592E %V 19 %X Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.
• Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo. Haack, Stephanie; Baiker, Sarah; Schlegel, Jan; Sauer, Markus; Sparwasser, Tim; Langenhorst, Daniela; Beyersdorf, Niklas. In European Journal of Immunology, 51(3), S. 738–741. John Wiley & Sons, Ltd, 2021.
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  Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.

  @article{haack2021superagonistic, abstract = {Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.}, author = {Haack, Stephanie and Baiker, Sarah and Schlegel, Jan and Sauer, Markus and Sparwasser, Tim and Langenhorst, Daniela and Beyersdorf, Niklas}, booktitle = {European Journal of Immunology}, journal = {European Journal of Immunology}, keywords = {sauer}, month = {03}, number = 3, pages = {738–741}, publisher = {John Wiley & Sons, Ltd}, title = {Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo}, volume = 51, year = 2021 }

  %0 Journal Article %1 haack2021superagonistic %A Haack, Stephanie %A Baiker, Sarah %A Schlegel, Jan %A Sauer, Markus %A Sparwasser, Tim %A Langenhorst, Daniela %A Beyersdorf, Niklas %B European Journal of Immunology %D 2021 %I John Wiley & Sons, Ltd %J European Journal of Immunology %N 3 %P 738--741 %R https://doi.org/10.1002/eji.202048803 %T Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo %U https://doi.org/10.1002/eji.202048803 %V 51 %X Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.
• Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures. Zwettler, Fabian U.; Reinhard, Sebastian; Sauer, Markus. In Expansion Microscopy for Cell Biology, Bd. 161, P. Guichard, V. Hamel (Hrsg.), S. 317–340. Academic Press, 2021.
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  This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.

  @inbook{zwettler2021chapter, abstract = {This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.}, author = {Zwettler, Fabian U. and Reinhard, Sebastian and Sauer, Markus}, booktitle = {Expansion Microscopy for Cell Biology}, editor = {Guichard, Paul and Hamel, Virginie}, keywords = {sauer}, pages = {317–340}, publisher = {Academic Press}, title = {Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures}, volume = 161, year = 2021 }

  %0 Book Section %1 zwettler2021chapter %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Sauer, Markus %B Expansion Microscopy for Cell Biology %D 2021 %E Guichard, Paul %E Hamel, Virginie %I Academic Press %P 317--340 %R https://doi.org/10.1016/bs.mcb.2020.05.004 %T Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures %U https://www.sciencedirect.com/science/article/pii/S0091679X20301126 %V 161 %X This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.
• Photoblueing of organic dyes can cause artifacts in super-resolution microscopy. Helmerich, Dominic A.; Beliu, Gerti; Matikonda, Siddharth S.; Schnermann, Martin J.; Sauer, Markus. In Nature Methods, 18(3), S. 253–257. 2021.
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  Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.

  @article{helmerich2021photoblueing, abstract = {Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Matikonda, Siddharth S. and Schnermann, Martin J. and Sauer, Markus}, journal = {Nature Methods}, keywords = {sauer}, number = 3, pages = {253–257}, title = {Photoblueing of organic dyes can cause artifacts in super-resolution microscopy}, volume = 18, year = 2021 }

  %0 Journal Article %1 helmerich2021photoblueing %A Helmerich, Dominic A. %A Beliu, Gerti %A Matikonda, Siddharth S. %A Schnermann, Martin J. %A Sauer, Markus %D 2021 %J Nature Methods %N 3 %P 253--257 %R 10.1038/s41592-021-01061-2 %T Photoblueing of organic dyes can cause artifacts in super-resolution microscopy %U https://doi.org/10.1038/s41592-021-01061-2 %V 18 %X Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.
• Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain. Beliu, Gerti; Altrichter, Steffen; Guixà-González, Ramon; Hemberger, Mareike; Brauer, Ina; Dahse, Anne-Kristin; Scholz, Nicole; Wieduwild, Robert; Kuhlemann, Alexander; Batebi, Hossein; Seufert, Florian; Pérez-Hernández, Guillermo; Hildebrand, Peter W.; Sauer, Markus; Langenhan, Tobias. In Molecular Cell, 81(5), S. 905–921.e5. 2021.
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  Summary Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

  @article{beliu2021tethered, abstract = {Summary Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.}, author = {Beliu, Gerti and Altrichter, Steffen and Guixà-González, Ramon and Hemberger, Mareike and Brauer, Ina and Dahse, Anne-Kristin and Scholz, Nicole and Wieduwild, Robert and Kuhlemann, Alexander and Batebi, Hossein and Seufert, Florian and Pérez-Hernández, Guillermo and Hildebrand, Peter W. and Sauer, Markus and Langenhan, Tobias}, journal = {Molecular Cell}, keywords = {sauer}, number = 5, pages = {905–921.e5}, title = {Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain}, volume = 81, year = 2021 }

  %0 Journal Article %1 beliu2021tethered %A Beliu, Gerti %A Altrichter, Steffen %A Guixà-González, Ramon %A Hemberger, Mareike %A Brauer, Ina %A Dahse, Anne-Kristin %A Scholz, Nicole %A Wieduwild, Robert %A Kuhlemann, Alexander %A Batebi, Hossein %A Seufert, Florian %A Pérez-Hernández, Guillermo %A Hildebrand, Peter W. %A Sauer, Markus %A Langenhan, Tobias %D 2021 %J Molecular Cell %N 5 %P 905--921.e5 %R https://doi.org/10.1016/j.molcel.2020.12.042 %T Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain %U https://www.sciencedirect.com/science/article/pii/S1097276520309631 %V 81 %X Summary Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.
• Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly. Jansch, Charline; Ziegler, Georg C.; Forero, Andrea; Gredy, Sina; Wäldchen, Sina; Vitale, Maria Rosaria; Svirin, Evgeniy; Zöller, Johanna E. M.; Waider, Jonas; Günther, Katharina; Edenhofer, Frank; Sauer, Markus; Wischmeyer, Erhard; Lesch, Klaus-Peter. In Journal of Neural Transmission, 128(2), S. 225–241. 2021.
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  Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.

  @article{jansch2021serotoninspecific, abstract = {Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (  42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.}, author = {Jansch, Charline and Ziegler, Georg C. and Forero, Andrea and Gredy, Sina and Wäldchen, Sina and Vitale, Maria Rosaria and Svirin, Evgeniy and Zöller, Johanna E. M. and Waider, Jonas and Günther, Katharina and Edenhofer, Frank and Sauer, Markus and Wischmeyer, Erhard and Lesch, Klaus-Peter}, journal = {Journal of Neural Transmission}, keywords = {sauer}, number = 2, pages = {225–241}, title = {Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly}, volume = 128, year = 2021 }

  %0 Journal Article %1 jansch2021serotoninspecific %A Jansch, Charline %A Ziegler, Georg C. %A Forero, Andrea %A Gredy, Sina %A Wäldchen, Sina %A Vitale, Maria Rosaria %A Svirin, Evgeniy %A Zöller, Johanna E. M. %A Waider, Jonas %A Günther, Katharina %A Edenhofer, Frank %A Sauer, Markus %A Wischmeyer, Erhard %A Lesch, Klaus-Peter %D 2021 %J Journal of Neural Transmission %N 2 %P 225--241 %R 10.1007/s00702-021-02303-5 %T Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly %U https://doi.org/10.1007/s00702-021-02303-5 %V 128 %X Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
• Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria. Peters, Simon; Kaiser, Lena; Fink, Julian; Schumacher, Fabian; Perschin, Veronika; Schlegel, Jan; Sauer, Markus; Stigloher, Christian; Kleuser, Burkhard; Seibel, Jürgen; Schubert-Unkmeir, Alexandra. In Scientific Reports, 11(1), S. 4300-. 2021.
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  Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.

  @article{peters2021clickcorrelative, abstract = {Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.}, author = {Peters, Simon and Kaiser, Lena and Fink, Julian and Schumacher, Fabian and Perschin, Veronika and Schlegel, Jan and Sauer, Markus and Stigloher, Christian and Kleuser, Burkhard and Seibel, Jürgen and Schubert-Unkmeir, Alexandra}, journal = {Scientific Reports}, keywords = {sauer}, number = 1, pages = {4300–}, title = {Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria}, volume = 11, year = 2021 }

  %0 Journal Article %1 peters2021clickcorrelative %A Peters, Simon %A Kaiser, Lena %A Fink, Julian %A Schumacher, Fabian %A Perschin, Veronika %A Schlegel, Jan %A Sauer, Markus %A Stigloher, Christian %A Kleuser, Burkhard %A Seibel, Jürgen %A Schubert-Unkmeir, Alexandra %D 2021 %J Scientific Reports %N 1 %P 4300-- %R 10.1038/s41598-021-83813-w %T Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria %U https://doi.org/10.1038/s41598-021-83813-w %V 11 %X Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
• Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis. Lehmann, Julian; Jørgensen, Morten E.; Fratz, Stefanie; Müller, Heike M.; Kusch, Jana; Scherzer, Sönke; Navarro-Retamal, Carlos; Mayer, Dominik; Böhm, Jennifer; Konrad, Kai R.; Terpitz, Ulrich; Dreyer, Ingo; Mueller, Thomas D.; Sauer, Markus; Hedrich, Rainer; Geiger, Dietmar; Maierhofer, Tobias. In Current Biology. 2021.
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  Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

  @article{lehmann2021acidosisinduced, abstract = {Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.}, author = {Lehmann, Julian and Jørgensen, Morten E. and Fratz, Stefanie and Müller, Heike M. and Kusch, Jana and Scherzer, Sönke and Navarro-Retamal, Carlos and Mayer, Dominik and Böhm, Jennifer and Konrad, Kai R. and Terpitz, Ulrich and Dreyer, Ingo and Mueller, Thomas D. and Sauer, Markus and Hedrich, Rainer and Geiger, Dietmar and Maierhofer, Tobias}, journal = {Current Biology}, keywords = {terpitz}, title = {Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis}, year = 2021 }

  %0 Journal Article %1 lehmann2021acidosisinduced %A Lehmann, Julian %A Jørgensen, Morten E. %A Fratz, Stefanie %A Müller, Heike M. %A Kusch, Jana %A Scherzer, Sönke %A Navarro-Retamal, Carlos %A Mayer, Dominik %A Böhm, Jennifer %A Konrad, Kai R. %A Terpitz, Ulrich %A Dreyer, Ingo %A Mueller, Thomas D. %A Sauer, Markus %A Hedrich, Rainer %A Geiger, Dietmar %A Maierhofer, Tobias %D 2021 %J Current Biology %R https://doi.org/10.1016/j.cub.2021.06.018 %T Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis %U https://www.sciencedirect.com/science/article/pii/S0960982221008137 %X Summary Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.
• Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab. García-Guerrero, Estefanía; Götz, Ralph; Doose, Sören; Sauer, Markus; Rodríguez-Gil, Alfonso; Nerreter, Thomas; Kortüm, K. Martin; Pérez-Simón, José A.; Einsele, Hermann; Hudecek, Michael; Danhof, Sophia. In Leukemia, 35(1), S. 201–214. 2021.
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  Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.

  @article{garciaguerrero2021upregulation, abstract = {Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.}, author = {García-Guerrero, Estefanía and Götz, Ralph and Doose, Sören and Sauer, Markus and Rodríguez-Gil, Alfonso and Nerreter, Thomas and Kortüm, K. Martin and Pérez-Simón, José A. and Einsele, Hermann and Hudecek, Michael and Danhof, Sophia}, journal = {Leukemia}, keywords = {sauer}, number = 1, pages = {201–214}, title = {Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab}, volume = 35, year = 2021 }

  %0 Journal Article %1 garciaguerrero2021upregulation %A García-Guerrero, Estefanía %A Götz, Ralph %A Doose, Sören %A Sauer, Markus %A Rodríguez-Gil, Alfonso %A Nerreter, Thomas %A Kortüm, K. Martin %A Pérez-Simón, José A. %A Einsele, Hermann %A Hudecek, Michael %A Danhof, Sophia %D 2021 %J Leukemia %N 1 %P 201--214 %R 10.1038/s41375-020-0840-y %T Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab %U https://doi.org/10.1038/s41375-020-0840-y %V 35 %X Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.
• Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections. Pauli, Martin; Paul, Mila M.; Proppert, Sven; Mrestani, Achmed; Sharifi, Marzieh; Repp, Felix; Kürzinger, Lydia; Kollmannsberger, Philip; Sauer, Markus; Heckmann, Manfred; Sirén, Anna-Leena. In Communications Biology, 4(1), S. 407-. 2021.
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  Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

  @article{pauli2021targeted, abstract = {Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.}, author = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and Kürzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sirén, Anna-Leena}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {407–}, title = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections}, volume = 4, year = 2021 }

  %0 Journal Article %1 pauli2021targeted %A Pauli, Martin %A Paul, Mila M. %A Proppert, Sven %A Mrestani, Achmed %A Sharifi, Marzieh %A Repp, Felix %A Kürzinger, Lydia %A Kollmannsberger, Philip %A Sauer, Markus %A Heckmann, Manfred %A Sirén, Anna-Leena %D 2021 %J Communications Biology %N 1 %P 407-- %R 10.1038/s42003-021-01939-z %T Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections %U https://doi.org/10.1038/s42003-021-01939-z %V 4 %X Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.
• Superresolution Microscopy of Sphingolipids. Schlegel, Jan; Sauer, Markus. In Lipid Rafts: Methods and Protocols, S. 303–311. Springer US, New York, NY, 2021.
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  This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.

  @inbook{schlegel2021superresolution, abstract = {This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.}, address = {New York, NY}, author = {Schlegel, Jan and Sauer, Markus}, booktitle = {Lipid Rafts: Methods and Protocols}, keywords = {sauer}, pages = {303–311}, publisher = {Springer US}, title = {Superresolution Microscopy of Sphingolipids}, year = 2021 }

  %0 Book Section %1 schlegel2021superresolution %A Schlegel, Jan %A Sauer, Markus %B Lipid Rafts: Methods and Protocols %C New York, NY %D 2021 %I Springer US %P 303--311 %R 10.1007/978-1-0716-0814-2_17 %T Superresolution Microscopy of Sphingolipids %U https://doi.org/10.1007/978-1-0716-0814-2_17 %X This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment. %@ 9781071608142
• Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy. Trinks, Nora; Reinhard, Sebastian; Drobny, Matthias; Heilig, Linda; Löffler, Jürgen; Sauer, Markus; Terpitz, Ulrich. In Communications Biology, 4(1), S. 1151-. 2021.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

  @article{trinks2021subdiffractionresolution, abstract = {Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.}, author = {Trinks, Nora and Reinhard, Sebastian and Drobny, Matthias and Heilig, Linda and Löffler, Jürgen and Sauer, Markus and Terpitz, Ulrich}, journal = {Communications Biology}, keywords = {terpitz}, number = 1, pages = {1151–}, title = {Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy}, volume = 4, year = 2021 }

  %0 Journal Article %1 trinks2021subdiffractionresolution %A Trinks, Nora %A Reinhard, Sebastian %A Drobny, Matthias %A Heilig, Linda %A Löffler, Jürgen %A Sauer, Markus %A Terpitz, Ulrich %D 2021 %J Communications Biology %N 1 %P 1151-- %R 10.1038/s42003-021-02669-y %T Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy %U https://doi.org/10.1038/s42003-021-02669-y %V 4 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
• A role for TASK2 channels in the human immunological synapse. Fernández-Orth, Juncal; Rolfes, Leoni; Gola, Lukas; Bittner, Stefan; Andronic, Joseph; Sukhorukov, Vladimir L.; Sisario, Dmitri; Landgraf, Peter; Dieterich, Daniela C.; Cerina, Manuela; Smalla, Karl-Heinz; Kähne, Thilo; Budde, Thomas; Kovac, Stjepana; Ruck, Tobias; Sauer, Markus; Meuth, Sven G. In European Journal of Immunology, 51(2), S. 342–353. John Wiley & Sons, Ltd, 2021.
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  Abstract The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

  @article{fernandezorth2021task2, abstract = {Abstract The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.}, author = {Fernández-Orth, Juncal and Rolfes, Leoni and Gola, Lukas and Bittner, Stefan and Andronic, Joseph and Sukhorukov, Vladimir L. and Sisario, Dmitri and Landgraf, Peter and Dieterich, Daniela C. and Cerina, Manuela and Smalla, Karl-Heinz and Kähne, Thilo and Budde, Thomas and Kovac, Stjepana and Ruck, Tobias and Sauer, Markus and Meuth, Sven G.}, booktitle = {European Journal of Immunology}, journal = {European Journal of Immunology}, keywords = {sauer}, month = {02}, number = 2, pages = {342–353}, publisher = {John Wiley & Sons, Ltd}, title = {A role for TASK2 channels in the human immunological synapse}, volume = 51, year = 2021 }

  %0 Journal Article %1 fernandezorth2021task2 %A Fernández-Orth, Juncal %A Rolfes, Leoni %A Gola, Lukas %A Bittner, Stefan %A Andronic, Joseph %A Sukhorukov, Vladimir L. %A Sisario, Dmitri %A Landgraf, Peter %A Dieterich, Daniela C. %A Cerina, Manuela %A Smalla, Karl-Heinz %A Kähne, Thilo %A Budde, Thomas %A Kovac, Stjepana %A Ruck, Tobias %A Sauer, Markus %A Meuth, Sven G. %B European Journal of Immunology %D 2021 %I John Wiley & Sons, Ltd %J European Journal of Immunology %N 2 %P 342--353 %R https://doi.org/10.1002/eji.201948269 %T A role for TASK2 channels in the human immunological synapse %U https://doi.org/10.1002/eji.201948269 %V 51 %X Abstract The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

• FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy. Memmel, Simon; Sisario, Dmitri; Zimmermann, Heiko; Sauer, Markus; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S.; Flentje, Michael. In BMC Bioinformatics, 21(1), S. 27-. 2020.
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  Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.

  @article{memmel2020focan, abstract = {Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.}, author = {Memmel, Simon and Sisario, Dmitri and Zimmermann, Heiko and Sauer, Markus and Sukhorukov, Vladimir L. and Djuzenova, Cholpon S. and Flentje, Michael}, journal = {BMC Bioinformatics}, keywords = {sauer}, number = 1, pages = {27–}, title = {FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy}, volume = 21, year = 2020 }

  %0 Journal Article %1 memmel2020focan %A Memmel, Simon %A Sisario, Dmitri %A Zimmermann, Heiko %A Sauer, Markus %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %A Flentje, Michael %D 2020 %J BMC Bioinformatics %N 1 %P 27-- %R 10.1186/s12859-020-3370-8 %T FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy %U https://doi.org/10.1186/s12859-020-3370-8 %V 21 %X Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.
• Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy. Götz, Ralph; Kunz, Tobias C.; Fink, Julian; Solger, Franziska; Schlegel, Jan; Seibel, Jürgen; Kozjak-Pavlovic, Vera; Rudel, Thomas; Sauer, Markus. In Nature Communications, 11(1), S. 6173-. 2020.
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  Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

  @article{gotz2020nanoscale, abstract = {Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.}, author = {Götz, Ralph and Kunz, Tobias C. and Fink, Julian and Solger, Franziska and Schlegel, Jan and Seibel, Jürgen and Kozjak-Pavlovic, Vera and Rudel, Thomas and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {6173–}, title = {Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy}, volume = 11, year = 2020 }

  %0 Journal Article %1 gotz2020nanoscale %A Götz, Ralph %A Kunz, Tobias C. %A Fink, Julian %A Solger, Franziska %A Schlegel, Jan %A Seibel, Jürgen %A Kozjak-Pavlovic, Vera %A Rudel, Thomas %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 6173-- %R 10.1038/s41467-020-19897-1 %T Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy %U https://doi.org/10.1038/s41467-020-19897-1 %V 11 %X Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.
• A Trojan Horse for live-cell super-resolution microscopy. Beliu, Gerti; Sauer, Markus. In Light: Science & Applications, 9(1), S. 2-. 2020.
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  New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.

  @article{beliu2020trojan, abstract = {New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.}, author = {Beliu, Gerti and Sauer, Markus}, journal = {Light: Science & Applications}, keywords = {sauer}, number = 1, pages = {2–}, title = {A Trojan Horse for live-cell super-resolution microscopy}, volume = 9, year = 2020 }

  %0 Journal Article %1 beliu2020trojan %A Beliu, Gerti %A Sauer, Markus %D 2020 %J Light: Science & Applications %N 1 %P 2-- %R 10.1038/s41377-019-0238-7 %T A Trojan Horse for live-cell super-resolution microscopy %U https://doi.org/10.1038/s41377-019-0238-7 %V 9 %X New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.
• ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein. Rozario, Ashley M.; Zwettler, Fabian; Duwé, Sam; Hargreaves, Riley B.; Brice, Aaron; Dedecker, Peter; Sauer, Markus; Moseley, Gregory W.; Whelan, Donna R.; Bell, Toby D. M. In Aust. J. Chem. 2020.
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  The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

  @article{rozario2020large, abstract = {The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.}, author = {Rozario, Ashley M. and Zwettler, Fabian and Duwé, Sam and Hargreaves, Riley B. and Brice, Aaron and Dedecker, Peter and Sauer, Markus and Moseley, Gregory W. and Whelan, Donna R. and Bell, Toby D. M.}, journal = {Aust. J. Chem.}, keywords = {sauer}, title = {‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein}, year = 2020 }

  %0 Journal Article %1 rozario2020large %A Rozario, Ashley M. %A Zwettler, Fabian %A Duwé, Sam %A Hargreaves, Riley B. %A Brice, Aaron %A Dedecker, Peter %A Sauer, Markus %A Moseley, Gregory W. %A Whelan, Donna R. %A Bell, Toby D. M. %D 2020 %J Aust. J. Chem. %T ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein %U https://doi.org/10.1071/CH19571 %X The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.
• Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus. Weiss, Esther; Schlegel, Jan; Terpitz, Ulrich; Weber, Michael; Linde, Jörg; Schmitt, Anna-Lena; Hünniger, Kerstin; Marischen, Lothar; Gamon, Florian; Bauer, Joachim; Löffler, Claudia; Kurzai, Oliver; Morton, Charles Oliver; Sauer, Markus; Einsele, Hermann; Loeffler, Juergen. In Frontiers in Immunology, 11. 2020.
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  Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

  @article{weiss2020reconstituting, abstract = {Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.}, author = {Weiss, Esther and Schlegel, Jan and Terpitz, Ulrich and Weber, Michael and Linde, Jörg and Schmitt, Anna-Lena and Hünniger, Kerstin and Marischen, Lothar and Gamon, Florian and Bauer, Joachim and Löffler, Claudia and Kurzai, Oliver and Morton, Charles Oliver and Sauer, Markus and Einsele, Hermann and Loeffler, Juergen}, journal = {Frontiers in Immunology}, keywords = {terpitz}, series = 2117, title = {Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus}, volume = 11, year = 2020 }

  %0 Journal Article %1 weiss2020reconstituting %A Weiss, Esther %A Schlegel, Jan %A Terpitz, Ulrich %A Weber, Michael %A Linde, Jörg %A Schmitt, Anna-Lena %A Hünniger, Kerstin %A Marischen, Lothar %A Gamon, Florian %A Bauer, Joachim %A Löffler, Claudia %A Kurzai, Oliver %A Morton, Charles Oliver %A Sauer, Markus %A Einsele, Hermann %A Loeffler, Juergen %B 2117 %D 2020 %J Frontiers in Immunology %T Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus %U https://www.frontiersin.org/article/10.3389/fimmu.2020.02117 %V 11 %X Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
• Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae. Kunz, Tobias C.; Götz, Ralph; Gao, Shiqiang; Sauer, Markus; Kozjak-Pavlovic, Vera. In Frontiers in Cell and Developmental Biology, 8, S. 617-. 2020.
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  Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.

  @article{kunz2020using, abstract = {Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.}, author = {Kunz, Tobias C. and Götz, Ralph and Gao, Shiqiang and Sauer, Markus and Kozjak-Pavlovic, Vera}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {617–}, title = {Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae}, volume = 8, year = 2020 }

  %0 Journal Article %1 kunz2020using %A Kunz, Tobias C. %A Götz, Ralph %A Gao, Shiqiang %A Sauer, Markus %A Kozjak-Pavlovic, Vera %D 2020 %J Frontiers in Cell and Developmental Biology %P 617-- %T Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae %U https://www.frontiersin.org/article/10.3389/fcell.2020.00617 %V 8 %X Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
• Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy. Zwettler, Fabian U.; Spindler, Marie-Christin; Reinhard, Sebastian; Klein, Teresa; Kurz, Andreas; Benavente, Ricardo; Sauer, Markus. In Nature Communications, 11(1), S. 3222-. 2020.
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  The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

  @article{zwettler2020tracking, abstract = {The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.}, author = {Zwettler, Fabian U. and Spindler, Marie-Christin and Reinhard, Sebastian and Klein, Teresa and Kurz, Andreas and Benavente, Ricardo and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3222–}, title = {Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy}, volume = 11, year = 2020 }

  %0 Journal Article %1 zwettler2020tracking %A Zwettler, Fabian U. %A Spindler, Marie-Christin %A Reinhard, Sebastian %A Klein, Teresa %A Kurz, Andreas %A Benavente, Ricardo %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3222-- %R 10.1038/s41467-020-17017-7 %T Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy %U https://doi.org/10.1038/s41467-020-17017-7 %V 11 %X The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.
• Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function. Forero, Andrea; Ku, Hsing-Ping; Malpartida, Ana Belén; Wäldchen, Sina; Alhama-Riba, Judit; Kulka, Christina; Aboagye, Benjamin; Norton, William H.J; Young, Andrew M.J; Ding, Yu-Qiang; Blum, Robert; Sauer, Markus; Rivero, Olga; Lesch, Klaus-Peter. In Neuropharmacology, 168, S. 108018-. 2020.
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  Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.

  @article{forero2020serotonin, abstract = {Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.}, author = {Forero, Andrea and Ku, Hsing-Ping and Malpartida, Ana Belén and Wäldchen, Sina and Alhama-Riba, Judit and Kulka, Christina and Aboagye, Benjamin and Norton, William H.J and Young, Andrew M.J and Ding, Yu-Qiang and Blum, Robert and Sauer, Markus and Rivero, Olga and Lesch, Klaus-Peter}, journal = {Neuropharmacology}, keywords = {sauer}, pages = {108018–}, title = {Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function}, volume = 168, year = 2020 }

  %0 Journal Article %1 forero2020serotonin %A Forero, Andrea %A Ku, Hsing-Ping %A Malpartida, Ana Belén %A Wäldchen, Sina %A Alhama-Riba, Judit %A Kulka, Christina %A Aboagye, Benjamin %A Norton, William H.J %A Young, Andrew M.J %A Ding, Yu-Qiang %A Blum, Robert %A Sauer, Markus %A Rivero, Olga %A Lesch, Klaus-Peter %D 2020 %J Neuropharmacology %P 108018-- %R https://doi.org/10.1016/j.neuropharm.2020.108018 %T Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function %U https://www.sciencedirect.com/science/article/pii/S0028390820300848 %V 168 %X Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.
• Expansion Microscopy for Cell Biology Analysis in Fungi. Götz, Ralph; Panzer, Sabine; Trinks, Nora; Eilts, Janna; Wagener, Johannes; Turrà, David; Di Pietro, Antonio; Sauer, Markus; Terpitz, Ulrich. In Frontiers in Microbiology, 11. 2020.
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  Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

  @article{gotz2020expansion, abstract = {Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.}, author = {Götz, Ralph and Panzer, Sabine and Trinks, Nora and Eilts, Janna and Wagener, Johannes and Turrà, David and Di Pietro, Antonio and Sauer, Markus and Terpitz, Ulrich}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, series = 574, title = {Expansion Microscopy for Cell Biology Analysis in Fungi}, volume = 11, year = 2020 }

  %0 Journal Article %1 gotz2020expansion %A Götz, Ralph %A Panzer, Sabine %A Trinks, Nora %A Eilts, Janna %A Wagener, Johannes %A Turrà, David %A Di Pietro, Antonio %A Sauer, Markus %A Terpitz, Ulrich %B 574 %D 2020 %J Frontiers in Microbiology %T Expansion Microscopy for Cell Biology Analysis in Fungi %U https://www.frontiersin.org/article/10.3389/fmicb.2020.00574 %V 11 %X Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
• Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM. Wäldchen, Felix; Schlegel, Jan; Götz, Ralph; Luciano, Michael; Schnermann, Martin; Doose, Sören; Sauer, Markus. In Nature Communications, 11(1), S. 887-. 2020.
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  The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

  @article{waldchen2020wholecell, abstract = {The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.}, author = {Wäldchen, Felix and Schlegel, Jan and Götz, Ralph and Luciano, Michael and Schnermann, Martin and Doose, Sören and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {887–}, title = {Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM}, volume = 11, year = 2020 }

  %0 Journal Article %1 waldchen2020wholecell %A Wäldchen, Felix %A Schlegel, Jan %A Götz, Ralph %A Luciano, Michael %A Schnermann, Martin %A Doose, Sören %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 887-- %R 10.1038/s41467-020-14731-0 %T Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM %U https://doi.org/10.1038/s41467-020-14731-0 %V 11 %X The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.
• Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy. Matikonda, Siddharth S.; Götz, Ralph; McLaughlin, Ryan; Sauer, Markus; Schnermann, Martin J. In Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods, Bd. 641, D. M. Chenoweth (Hrsg.), S. 225–244. Academic Press, 2020.
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  Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).

  @inbook{matikonda2020chapter, abstract = {Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).}, author = {Matikonda, Siddharth S. and Götz, Ralph and McLaughlin, Ryan and Sauer, Markus and Schnermann, Martin J.}, booktitle = {Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods}, editor = {Chenoweth, David M.}, keywords = {sauer}, pages = {225–244}, publisher = {Academic Press}, title = {Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy}, volume = 641, year = 2020 }

  %0 Book Section %1 matikonda2020chapter %A Matikonda, Siddharth S. %A Götz, Ralph %A McLaughlin, Ryan %A Sauer, Markus %A Schnermann, Martin J. %B Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods %D 2020 %E Chenoweth, David M. %I Academic Press %P 225--244 %R https://doi.org/10.1016/bs.mie.2020.04.042 %T Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy %U https://www.sciencedirect.com/science/article/pii/S0076687920301701 %V 641 %X Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).
• Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager. Kouhestani, Dina; Geis, Maria; Alsouri, Saed; Bumm, Thomas G. P.; Einsele, Hermann; Sauer, Markus; Stuhler, Gernot. In Cellular & Molecular Immunology. 2020.
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  @article{kouhestani2020variant, author = {Kouhestani, Dina and Geis, Maria and Alsouri, Saed and Bumm, Thomas G. P. and Einsele, Hermann and Sauer, Markus and Stuhler, Gernot}, journal = {Cellular & Molecular Immunology}, keywords = {sauer}, title = {Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager}, year = 2020 }

  %0 Journal Article %1 kouhestani2020variant %A Kouhestani, Dina %A Geis, Maria %A Alsouri, Saed %A Bumm, Thomas G. P. %A Einsele, Hermann %A Sauer, Markus %A Stuhler, Gernot %D 2020 %J Cellular & Molecular Immunology %R 10.1038/s41423-020-0507-7 %T Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager %U https://doi.org/10.1038/s41423-020-0507-7
• Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Zwettler, Fabian U.; Reinhard, Sebastian; Gambarotto, Davide; Bell, Toby D. M.; Hamel, Virginie; Guichard, Paul; Sauer, Markus. In Nature Communications, 11(1), S. 3388-. 2020.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

  @article{zwettler2020molecular, abstract = {Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.}, author = {Zwettler, Fabian U. and Reinhard, Sebastian and Gambarotto, Davide and Bell, Toby D. M. and Hamel, Virginie and Guichard, Paul and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3388–}, title = {Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)}, volume = 11, year = 2020 }

  %0 Journal Article %1 zwettler2020molecular %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Gambarotto, Davide %A Bell, Toby D. M. %A Hamel, Virginie %A Guichard, Paul %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3388-- %R 10.1038/s41467-020-17086-8 %T Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM) %U https://doi.org/10.1038/s41467-020-17086-8 %V 11 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.
• Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones. Siddig, Sana; Aufmkolk, Sarah; Doose, Sören; Jobin, Marie-Lise; Werner, Christian; Sauer, Markus; Calebiro, Davide. In Sci Adv, 6(16), S. eaay7193-. 2020.
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  G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.

  @article{siddig2020superresolution, abstract = {G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.}, author = {Siddig, Sana and Aufmkolk, Sarah and Doose, Sören and Jobin, Marie-Lise and Werner, Christian and Sauer, Markus and Calebiro, Davide}, journal = {Sci Adv}, keywords = {home}, month = {04}, number = 16, pages = {eaay7193–}, title = {Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones}, volume = 6, year = 2020 }

  %0 Journal Article %1 siddig2020superresolution %A Siddig, Sana %A Aufmkolk, Sarah %A Doose, Sören %A Jobin, Marie-Lise %A Werner, Christian %A Sauer, Markus %A Calebiro, Davide %D 2020 %J Sci Adv %N 16 %P eaay7193-- %R 10.1126/sciadv.aay7193 %T Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones %U http://advances.sciencemag.org/content/6/16/eaay7193.abstract %V 6 %X G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.
• Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer. Helmerich, Dominic A.; Beliu, Gerti; Sauer, Markus. In ACS Nano. American Chemical Society, 2020.
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  The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.

  @article{helmerich2020multiplelabeled, abstract = {The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Sauer, Markus}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = {08}, publisher = {American Chemical Society}, title = {Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer}, year = 2020 }

  %0 Journal Article %1 helmerich2020multiplelabeled %A Helmerich, Dominic A. %A Beliu, Gerti %A Sauer, Markus %B ACS Nano %D 2020 %I American Chemical Society %J ACS Nano %R 10.1021/acsnano.0c06099 %T Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer %U https://doi.org/10.1021/acsnano.0c06099 %X The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.
• Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning. Andreska, Thomas; Rauskolb, Stefanie; Schukraft, Nina; Lüningschrör, Patrick; Sasi, Manju; Signoret-Genest, Jeremy; Behringer, Marcus; Blum, Robert; Sauer, Markus; Tovote, Philip; Sendtner, Michael. In J. Neurosci., 40(33), S. 6289-. 2020.
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  Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.

  @article{andreska2020induction, abstract = {Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.}, author = {Andreska, Thomas and Rauskolb, Stefanie and Schukraft, Nina and Lüningschrör, Patrick and Sasi, Manju and Signoret-Genest, Jeremy and Behringer, Marcus and Blum, Robert and Sauer, Markus and Tovote, Philip and Sendtner, Michael}, journal = {J. Neurosci.}, keywords = {sauer}, month = {08}, number = 33, pages = {6289–}, title = {Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning}, volume = 40, year = 2020 }

  %0 Journal Article %1 andreska2020induction %A Andreska, Thomas %A Rauskolb, Stefanie %A Schukraft, Nina %A Lüningschrör, Patrick %A Sasi, Manju %A Signoret-Genest, Jeremy %A Behringer, Marcus %A Blum, Robert %A Sauer, Markus %A Tovote, Philip %A Sendtner, Michael %D 2020 %J J. Neurosci. %N 33 %P 6289-- %R 10.1523/JNEUROSCI.0288-20.2020 %T Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning %U http://www.jneurosci.org/content/40/33/6289.abstract %V 40 %X Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.
• Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy. Thiele, Jan Christoph; Helmerich, Dominic A.; Oleksiievets, Nazar; Tsukanov, Roman; Butkevich, Eugenia; Sauer, Markus; Nevskyi, Oleksii; Enderlein, Jörg. In ACS Nano, 14(10), S. 14190–14200. American Chemical Society, 2020.
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  Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

  @article{thiele2020confocal, abstract = {Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.}, author = {Thiele, Jan Christoph and Helmerich, Dominic A. and Oleksiievets, Nazar and Tsukanov, Roman and Butkevich, Eugenia and Sauer, Markus and Nevskyi, Oleksii and Enderlein, Jörg}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = 10, number = 10, pages = {14190–14200}, publisher = {American Chemical Society}, title = {Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy}, volume = 14, year = 2020 }

  %0 Journal Article %1 thiele2020confocal %A Thiele, Jan Christoph %A Helmerich, Dominic A. %A Oleksiievets, Nazar %A Tsukanov, Roman %A Butkevich, Eugenia %A Sauer, Markus %A Nevskyi, Oleksii %A Enderlein, Jörg %B ACS Nano %D 2020 %I American Chemical Society %J ACS Nano %N 10 %P 14190--14200 %R 10.1021/acsnano.0c07322 %T Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy %U https://doi.org/10.1021/acsnano.0c07322 %V 14 %X Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.
• BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation. Volz, Julia; Kusch, Charly; Beck, Sarah; Popp, Michael; Vögtle, Timo; Meub, Mara; Scheller, Inga; Heil, Hannah S.; Preu, Julia; Schuhmann, Michael K.; Hemmen, Katherina; Premsler, Thomas; Sickmann, Albert; Heinze, Katrin G.; Stegner, David; Stoll, Guido; Braun, Attila; Sauer, Markus; Nieswandt, Bernhard. In The Journal of Clinical Investigation, 130(11), S. 6064–6079. The American Society for Clinical Investigation, 2020.
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  Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

  @article{volz2020orchestrates, abstract = {Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.}, author = {Volz, Julia and Kusch, Charly and Beck, Sarah and Popp, Michael and Vögtle, Timo and Meub, Mara and Scheller, Inga and Heil, Hannah S. and Preu, Julia and Schuhmann, Michael K. and Hemmen, Katherina and Premsler, Thomas and Sickmann, Albert and Heinze, Katrin G. and Stegner, David and Stoll, Guido and Braun, Attila and Sauer, Markus and Nieswandt, Bernhard}, journal = {The Journal of Clinical Investigation}, keywords = {sauer}, month = 11, number = 11, pages = {6064–6079}, publisher = {The American Society for Clinical Investigation}, title = {BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation}, volume = 130, year = 2020 }

  %0 Journal Article %1 volz2020orchestrates %A Volz, Julia %A Kusch, Charly %A Beck, Sarah %A Popp, Michael %A Vögtle, Timo %A Meub, Mara %A Scheller, Inga %A Heil, Hannah S. %A Preu, Julia %A Schuhmann, Michael K. %A Hemmen, Katherina %A Premsler, Thomas %A Sickmann, Albert %A Heinze, Katrin G. %A Stegner, David %A Stoll, Guido %A Braun, Attila %A Sauer, Markus %A Nieswandt, Bernhard %D 2020 %I The American Society for Clinical Investigation %J The Journal of Clinical Investigation %N 11 %P 6064--6079 %R 10.1172/JCI136457 %T BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation %U https://doi.org/10.1172/JCI136457 %V 130 %X Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

• Imaging cellular ultrastructures using expansion microscopy (U-ExM). Gambarotto, Davide; Zwettler, Fabian U.; Le Guennec, Maeva; Schmidt-Cernohorska, Marketa; Fortun, Denis; Borgers, Susanne; Heine, Jörn; Schloetel, Jan-Gero; Reuss, Matthias; Unser, Michael; Boyden, Edward S.; Sauer, Markus; Hamel, Virginie; Guichard, Paul. In Nature Methods, 16(1), S. 71–74. 2019.
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  Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

  @article{gambarotto2019imaging, abstract = {Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.}, author = {Gambarotto, Davide and Zwettler, Fabian U. and Le Guennec, Maeva and Schmidt-Cernohorska, Marketa and Fortun, Denis and Borgers, Susanne and Heine, Jörn and Schloetel, Jan-Gero and Reuss, Matthias and Unser, Michael and Boyden, Edward S. and Sauer, Markus and Hamel, Virginie and Guichard, Paul}, journal = {Nature Methods}, keywords = {sauer}, number = 1, pages = {71–74}, title = {Imaging cellular ultrastructures using expansion microscopy (U-ExM)}, volume = 16, year = 2019 }

  %0 Journal Article %1 gambarotto2019imaging %A Gambarotto, Davide %A Zwettler, Fabian U. %A Le Guennec, Maeva %A Schmidt-Cernohorska, Marketa %A Fortun, Denis %A Borgers, Susanne %A Heine, Jörn %A Schloetel, Jan-Gero %A Reuss, Matthias %A Unser, Michael %A Boyden, Edward S. %A Sauer, Markus %A Hamel, Virginie %A Guichard, Paul %D 2019 %J Nature Methods %N 1 %P 71--74 %R 10.1038/s41592-018-0238-1 %T Imaging cellular ultrastructures using expansion microscopy (U-ExM) %U https://doi.org/10.1038/s41592-018-0238-1 %V 16 %X Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
• Super-resolution microscopy demystified. Schermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C. In Nature Cell Biology, 21(1), S. 72–84. 2019.
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  Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

  @article{schermelleh2019superresolution, abstract = {Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.}, author = {Schermelleh, Lothar and Ferrand, Alexia and Huser, Thomas and Eggeling, Christian and Sauer, Markus and Biehlmaier, Oliver and Drummen, Gregor P. C.}, journal = {Nature Cell Biology}, keywords = {home}, number = 1, pages = {72–84}, title = {Super-resolution microscopy demystified}, volume = 21, year = 2019 }

  %0 Journal Article %1 schermelleh2019superresolution %A Schermelleh, Lothar %A Ferrand, Alexia %A Huser, Thomas %A Eggeling, Christian %A Sauer, Markus %A Biehlmaier, Oliver %A Drummen, Gregor P. C. %D 2019 %J Nature Cell Biology %N 1 %P 72--84 %R 10.1038/s41556-018-0251-8 %T Super-resolution microscopy demystified %U https://doi.org/10.1038/s41556-018-0251-8 %V 21 %X Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
• Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T. Nerreter, Thomas; Letschert, Sebastian; Götz, Ralph; Doose, Sören; Danhof, Sophia; Einsele, Hermann; Sauer, Markus; Hudecek, Michael. In Nature Communications, 10(1), S. 3137-. 2019.
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  Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.

  @article{nerreter2019superresolution, abstract = {Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.}, author = {Nerreter, Thomas and Letschert, Sebastian and Götz, Ralph and Doose, Sören and Danhof, Sophia and Einsele, Hermann and Sauer, Markus and Hudecek, Michael}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3137–}, title = {Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T}, volume = 10, year = 2019 }

  %0 Journal Article %1 nerreter2019superresolution %A Nerreter, Thomas %A Letschert, Sebastian %A Götz, Ralph %A Doose, Sören %A Danhof, Sophia %A Einsele, Hermann %A Sauer, Markus %A Hudecek, Michael %D 2019 %J Nature Communications %N 1 %P 3137-- %R 10.1038/s41467-019-10948-w %T Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T %U https://doi.org/10.1038/s41467-019-10948-w %V 10 %X Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
• Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Beliu, Gerti; Kurz, Andreas J.; Kuhlemann, Alexander C.; Behringer-Pliess, Lisa; Meub, Mara; Wolf, Natalia; Seibel, Jürgen; Shi, Zhen-Dan; Schnermann, Martin; Grimm, Jonathan B.; Lavis, Luke D.; Doose, Sören; Sauer, Markus. In Communications Biology, 2(1), S. 261-. 2019.
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  Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

  @article{beliu2019bioorthogonal, abstract = {Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.}, author = {Beliu, Gerti and Kurz, Andreas J. and Kuhlemann, Alexander C. and Behringer-Pliess, Lisa and Meub, Mara and Wolf, Natalia and Seibel, Jürgen and Shi, Zhen-Dan and Schnermann, Martin and Grimm, Jonathan B. and Lavis, Luke D. and Doose, Sören and Sauer, Markus}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {261–}, title = {Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy}, volume = 2, year = 2019 }

  %0 Journal Article %1 beliu2019bioorthogonal %A Beliu, Gerti %A Kurz, Andreas J. %A Kuhlemann, Alexander C. %A Behringer-Pliess, Lisa %A Meub, Mara %A Wolf, Natalia %A Seibel, Jürgen %A Shi, Zhen-Dan %A Schnermann, Martin %A Grimm, Jonathan B. %A Lavis, Luke D. %A Doose, Sören %A Sauer, Markus %D 2019 %J Communications Biology %N 1 %P 261-- %R 10.1038/s42003-019-0518-z %T Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy %U https://doi.org/10.1038/s42003-019-0518-z %V 2 %X Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
• FSP1 is a glutathione-independent ferroptosis suppressor. Doll, Sebastian; Freitas, Florencio Porto; Shah, Ron; Aldrovandi, Maceler; da Silva, Milene Costa; Ingold, Irina; Grocin, Andrea Goya; Xavier da Silva, Thamara Nishida; Panzilius, Elena; Scheel, Christina H.; Mourão, André; Buday, Katalin; Sato, Mami; Wanninger, Jonas; Vignane, Thibaut; Mohana, Vaishnavi; Rehberg, Markus; Flatley, Andrew; Schepers, Aloys; Kurz, Andreas; White, Daniel; Sauer, Markus; Sattler, Michael; Tate, Edward William; Schmitz, Werner; Schulze, Almut; O’Donnell, Valerie; Proneth, Bettina; Popowicz, Grzegorz M.; Pratt, Derek A.; Angeli, José Pedro Friedmann; Conrad, Marcus. In Nature, 575(7784), S. 693–698. 2019.
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  Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

  @article{doll2019glutathioneindependent, abstract = {Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.}, author = {Doll, Sebastian and Freitas, Florencio Porto and Shah, Ron and Aldrovandi, Maceler and da Silva, Milene Costa and Ingold, Irina and Grocin, Andrea Goya and Xavier da Silva, Thamara Nishida and Panzilius, Elena and Scheel, Christina H. and Mourão, André and Buday, Katalin and Sato, Mami and Wanninger, Jonas and Vignane, Thibaut and Mohana, Vaishnavi and Rehberg, Markus and Flatley, Andrew and Schepers, Aloys and Kurz, Andreas and White, Daniel and Sauer, Markus and Sattler, Michael and Tate, Edward William and Schmitz, Werner and Schulze, Almut and O’Donnell, Valerie and Proneth, Bettina and Popowicz, Grzegorz M. and Pratt, Derek A. and Angeli, José Pedro Friedmann and Conrad, Marcus}, journal = {Nature}, keywords = {sauer}, number = 7784, pages = {693–698}, title = {FSP1 is a glutathione-independent ferroptosis suppressor}, volume = 575, year = 2019 }

  %0 Journal Article %1 doll2019glutathioneindependent %A Doll, Sebastian %A Freitas, Florencio Porto %A Shah, Ron %A Aldrovandi, Maceler %A da Silva, Milene Costa %A Ingold, Irina %A Grocin, Andrea Goya %A Xavier da Silva, Thamara Nishida %A Panzilius, Elena %A Scheel, Christina H. %A Mourão, André %A Buday, Katalin %A Sato, Mami %A Wanninger, Jonas %A Vignane, Thibaut %A Mohana, Vaishnavi %A Rehberg, Markus %A Flatley, Andrew %A Schepers, Aloys %A Kurz, Andreas %A White, Daniel %A Sauer, Markus %A Sattler, Michael %A Tate, Edward William %A Schmitz, Werner %A Schulze, Almut %A O’Donnell, Valerie %A Proneth, Bettina %A Popowicz, Grzegorz M. %A Pratt, Derek A. %A Angeli, José Pedro Friedmann %A Conrad, Marcus %D 2019 %J Nature %N 7784 %P 693--698 %R 10.1038/s41586-019-1707-0 %T FSP1 is a glutathione-independent ferroptosis suppressor %U https://doi.org/10.1038/s41586-019-1707-0 %V 575 %X Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.
• Platelet lamellipodium formation is not required for thrombus formation and stability. Schurr, Yvonne; Sperr, Andreas; Volz, Julia; Beck, Sarah; Reil, Lucy; Kusch, Charly; Eiring, Patrick; Bryson, Sheila; Sauer, Markus; Nieswandt, Bernhard; Machesky, Laura; Bender, Markus. In Blood, 134, S. 2318–2329. 2019.
  • [ Abstract ]
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  During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

  @article{schurr2019platelet, abstract = {During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.}, author = {Schurr, Yvonne and Sperr, Andreas and Volz, Julia and Beck, Sarah and Reil, Lucy and Kusch, Charly and Eiring, Patrick and Bryson, Sheila and Sauer, Markus and Nieswandt, Bernhard and Machesky, Laura and Bender, Markus}, booktitle = {Blood}, journal = {Blood}, keywords = {sauer}, pages = {2318-2329}, series = 25, title = {Platelet lamellipodium formation is not required for thrombus formation and stability}, volume = 134, year = 2019 }

  %0 Journal Article %1 schurr2019platelet %A Schurr, Yvonne %A Sperr, Andreas %A Volz, Julia %A Beck, Sarah %A Reil, Lucy %A Kusch, Charly %A Eiring, Patrick %A Bryson, Sheila %A Sauer, Markus %A Nieswandt, Bernhard %A Machesky, Laura %A Bender, Markus %B Blood %D 2019 %J Blood %P 2318-2329 %T Platelet lamellipodium formation is not required for thrombus formation and stability %U https://doi.org/10.1182/blood.2019002105 %V 134 %X During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.
• Generation of site-distinct N-glycan variants for in vitro bioactivity testing. Brühlmann, David; Vuillemin, Thomas; Satwekar, Abhijeet; Galano, Eugenio; Palmese, Angelo; D’Angelo, Alessandra; Manco, Zeynep; Souquet, Jonathan; Broly, Hervé; Sauer, Markus; Hemberger, Jürgen; Jordan, Martin. In Biotechnology and Bioengineering, 116(5), S. 1017–1028. John Wiley & Sons, Ltd, 2019.
  • [ Abstract ]
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  Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

  @article{bruhlmann2019generation, abstract = {Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.}, author = {Brühlmann, David and Vuillemin, Thomas and Satwekar, Abhijeet and Galano, Eugenio and Palmese, Angelo and D'Angelo, Alessandra and Manco, Zeynep and Souquet, Jonathan and Broly, Hervé and Sauer, Markus and Hemberger, Jürgen and Jordan, Martin}, booktitle = {Biotechnology and Bioengineering}, journal = {Biotechnology and Bioengineering}, keywords = {sauer}, month = {05}, number = 5, pages = {1017–1028}, publisher = {John Wiley & Sons, Ltd}, title = {Generation of site-distinct N-glycan variants for in vitro bioactivity testing}, volume = 116, year = 2019 }

  %0 Journal Article %1 bruhlmann2019generation %A Brühlmann, David %A Vuillemin, Thomas %A Satwekar, Abhijeet %A Galano, Eugenio %A Palmese, Angelo %A D'Angelo, Alessandra %A Manco, Zeynep %A Souquet, Jonathan %A Broly, Hervé %A Sauer, Markus %A Hemberger, Jürgen %A Jordan, Martin %B Biotechnology and Bioengineering %D 2019 %I John Wiley & Sons, Ltd %J Biotechnology and Bioengineering %N 5 %P 1017--1028 %R 10.1002/bit.26930 %T Generation of site-distinct N-glycan variants for in vitro bioactivity testing %U https://doi.org/10.1002/bit.26930 %V 116 %X Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.
• Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium. Derakhshani, Shaghayegh; Kurz, Andreas; Japtok, Lukasz; Schumacher, Fabian; Pilgram, Lisa; Steinke, Maria; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle; Avota, Elita. In Frontiers in Immunology, 10, S. 1294-. 2019.
  • [ Abstract ]
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  Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.

  @article{derakhshani2019measles, abstract = {Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.}, author = {Derakhshani, Shaghayegh and Kurz, Andreas and Japtok, Lukasz and Schumacher, Fabian and Pilgram, Lisa and Steinke, Maria and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, journal = {Frontiers in Immunology}, keywords = {sauer}, pages = {1294–}, title = {Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium}, volume = 10, year = 2019 }

  %0 Journal Article %1 derakhshani2019measles %A Derakhshani, Shaghayegh %A Kurz, Andreas %A Japtok, Lukasz %A Schumacher, Fabian %A Pilgram, Lisa %A Steinke, Maria %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2019 %J Frontiers in Immunology %P 1294-- %T Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium %U https://www.frontiersin.org/article/10.3389/fimmu.2019.01294 %V 10 %X Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.
• Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites. Schlegel, Jan; Peters, Simon; Doose, Sören; Schubert-Unkmeir, Alexandra; Sauer, Markus. In Frontiers in Cell and Developmental Biology, 7, S. 194-. 2019.
  • [ Abstract ]
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  Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.

  @article{schlegel2019superresolution, abstract = {Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.}, author = {Schlegel, Jan and Peters, Simon and Doose, Sören and Schubert-Unkmeir, Alexandra and Sauer, Markus}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {194–}, title = {Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites}, volume = 7, year = 2019 }

  %0 Journal Article %1 schlegel2019superresolution %A Schlegel, Jan %A Peters, Simon %A Doose, Sören %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2019 %J Frontiers in Cell and Developmental Biology %P 194-- %T Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites %U https://www.frontiersin.org/article/10.3389/fcell.2019.00194 %V 7 %X Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.
• Registration and Visualization of Correlative Super-Resolution Microscopy Data. Reinhard, Sebastian; Aufmkolk, Sarah; Sauer, Markus; Doose, Sören. In Biophysical Journal, 116(11), S. 2073–2078. 2019.
  • [ Abstract ]
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  We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.

  @article{reinhard2019registration, abstract = {We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.}, author = {Reinhard, Sebastian and Aufmkolk, Sarah and Sauer, Markus and Doose, Sören}, journal = {Biophysical Journal}, keywords = {sauer}, number = 11, pages = {2073–2078}, title = {Registration and Visualization of Correlative Super-Resolution Microscopy Data}, volume = 116, year = 2019 }

  %0 Journal Article %1 reinhard2019registration %A Reinhard, Sebastian %A Aufmkolk, Sarah %A Sauer, Markus %A Doose, Sören %D 2019 %J Biophysical Journal %N 11 %P 2073--2078 %R https://doi.org/10.1016/j.bpj.2019.04.029 %T Registration and Visualization of Correlative Super-Resolution Microscopy Data %U http://www.sciencedirect.com/science/article/pii/S000634951930373X %V 116 %X We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
• Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells. Zilkowski, Ilona; Ziouti, Fani; Schulze, Andres; Hauck, Stefanie; Schmidt, Stefanie; Mainz, Laura; Sauer, Markus; Albrecht, Krystyna; Jundt, Franziska; Groll, Jürgen. In Biomacromolecules, 20(2), S. 916–926. American Chemical Society, 2019.
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  Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.

  @article{zilkowski2019nanogels, abstract = {Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.}, author = {Zilkowski, Ilona and Ziouti, Fani and Schulze, Andres and Hauck, Stefanie and Schmidt, Stefanie and Mainz, Laura and Sauer, Markus and Albrecht, Krystyna and Jundt, Franziska and Groll, Jürgen}, booktitle = {Biomacromolecules}, journal = {Biomacromolecules}, keywords = {sauer}, month = {02}, number = 2, pages = {916–926}, publisher = {American Chemical Society}, title = {Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells}, volume = 20, year = 2019 }

  %0 Journal Article %1 zilkowski2019nanogels %A Zilkowski, Ilona %A Ziouti, Fani %A Schulze, Andres %A Hauck, Stefanie %A Schmidt, Stefanie %A Mainz, Laura %A Sauer, Markus %A Albrecht, Krystyna %A Jundt, Franziska %A Groll, Jürgen %B Biomacromolecules %D 2019 %I American Chemical Society %J Biomacromolecules %N 2 %P 916--926 %R 10.1021/acs.biomac.8b01553 %T Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells %U https://doi.org/10.1021/acs.biomac.8b01553 %V 20 %X Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.
• Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels. Peters, Simon; Schlegel, Jan; Becam, Jérôme; Avota, Elita; Sauer, Markus; Schubert-Unkmeir, Alexandra. In Infect. Immun., 87(8), A. J. Bäumler (Hrsg.), S. e00410–19. 2019.
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  Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

  @article{peters2019ltspan, abstract = {Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.}, author = {Peters, Simon and Schlegel, Jan and Becam, Jérôme and Avota, Elita and Sauer, Markus and Schubert-Unkmeir, Alexandra}, editor = {Bäumler, Andreas J.}, journal = {Infect. Immun.}, keywords = {sauer}, month = {08}, number = 8, pages = {e00410-19–}, title = {Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels}, volume = 87, year = 2019 }

  %0 Journal Article %1 peters2019ltspan %A Peters, Simon %A Schlegel, Jan %A Becam, Jérôme %A Avota, Elita %A Sauer, Markus %A Schubert-Unkmeir, Alexandra %D 2019 %E Bäumler, Andreas J. %J Infect. Immun. %N 8 %P e00410-19-- %R 10.1128/IAI.00410-19 %T Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels %U http://iai.asm.org/content/87/8/e00410-19.abstract %V 87 %X Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.
• Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy. Kunz, Tobias C.; Götz, Ralph; Sauer, Markus; Rudel, Thomas. In Frontiers in Cellular and Infection Microbiology, 9, S. 276-. 2019.
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  Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

  @article{kunz2019detection, abstract = {Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.}, author = {Kunz, Tobias C. and Götz, Ralph and Sauer, Markus and Rudel, Thomas}, journal = {Frontiers in Cellular and Infection Microbiology}, keywords = {sauer}, pages = {276–}, title = {Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy}, volume = 9, year = 2019 }

  %0 Journal Article %1 kunz2019detection %A Kunz, Tobias C. %A Götz, Ralph %A Sauer, Markus %A Rudel, Thomas %D 2019 %J Frontiers in Cellular and Infection Microbiology %P 276-- %T Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy %U https://www.frontiersin.org/article/10.3389/fcimb.2019.00276 %V 9 %X Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

• Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging. Neubert, Franziska; Beliu, Gerti; Terpitz, Ulrich; Werner, Christian; Geis, Christian; Sauer, Markus; Doose, Sören. In Angewandte Chemie International Edition, 57(50), S. 16364–16369. John Wiley & Sons, Ltd, 2018.
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  Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

  @article{neubert2018bioorthogonal, abstract = {Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.}, author = {Neubert, Franziska and Beliu, Gerti and Terpitz, Ulrich and Werner, Christian and Geis, Christian and Sauer, Markus and Doose, Sören}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {terpitz}, month = 10, number = 50, pages = {16364–16369}, publisher = {John Wiley & Sons, Ltd}, title = {Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging}, volume = 57, year = 2018 }

  %0 Journal Article %1 neubert2018bioorthogonal %A Neubert, Franziska %A Beliu, Gerti %A Terpitz, Ulrich %A Werner, Christian %A Geis, Christian %A Sauer, Markus %A Doose, Sören %B Angewandte Chemie International Edition %D 2018 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 50 %P 16364--16369 %R 10.1002/anie.201808951 %T Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging %U https://doi.org/10.1002/anie.201808951 %V 57 %X Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.
• Nanostructure of DNA repair foci revealed by superresolution microscopy. Sisario, Dmitri; Memmel, Simon; Doose, Sören; Neubauer, Julia; Zimmermann, Heiko; Flentje, Michael; Djuzenova, Cholpon S.; Sauer, Markus; Sukhorukov, Vladimir L. In The FASEB Journal, S. fj.201701435-. Federation of American Societies for Experimental Biology, 2018.
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  Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

  @article{sisario2018nanostructure, abstract = {Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.}, author = {Sisario, Dmitri and Memmel, Simon and Doose, Sören and Neubauer, Julia and Zimmermann, Heiko and Flentje, Michael and Djuzenova, Cholpon S. and Sauer, Markus and Sukhorukov, Vladimir L.}, booktitle = {The FASEB Journal}, journal = {The FASEB Journal}, keywords = {sauer}, month = {06}, pages = {fj.201701435–}, publisher = {Federation of American Societies for Experimental Biology}, title = {Nanostructure of DNA repair foci revealed by superresolution microscopy}, year = 2018 }

  %0 Journal Article %1 sisario2018nanostructure %A Sisario, Dmitri %A Memmel, Simon %A Doose, Sören %A Neubauer, Julia %A Zimmermann, Heiko %A Flentje, Michael %A Djuzenova, Cholpon S. %A Sauer, Markus %A Sukhorukov, Vladimir L. %B The FASEB Journal %D 2018 %I Federation of American Societies for Experimental Biology %J The FASEB Journal %P fj.201701435-- %R 10.1096/fj.201701435 %T Nanostructure of DNA repair foci revealed by superresolution microscopy %U https://doi.org/10.1096/fj.201701435 %X Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
• Sharpening emitter localization in front of a tuned mirror. Heil, Hannah S.; Schreiber, Benjamin; Götz, Ralph; Emmerling, Monika; Dabauvalle, Marie-Christine; Krohne, Georg; Höfling, Sven; Kamp, Martin; Sauer, Markus; Heinze, Katrin G. In Light: Science & Applications, 7(1), S. 99-. 2018.
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  Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.

  @article{heil2018sharpening, abstract = {Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.}, author = {Heil, Hannah S. and Schreiber, Benjamin and Götz, Ralph and Emmerling, Monika and Dabauvalle, Marie-Christine and Krohne, Georg and Höfling, Sven and Kamp, Martin and Sauer, Markus and Heinze, Katrin G.}, journal = {Light: Science & Applications}, keywords = {sauer}, number = 1, pages = {99–}, title = {Sharpening emitter localization in front of a tuned mirror}, volume = 7, year = 2018 }

  %0 Journal Article %1 heil2018sharpening %A Heil, Hannah S. %A Schreiber, Benjamin %A Götz, Ralph %A Emmerling, Monika %A Dabauvalle, Marie-Christine %A Krohne, Georg %A Höfling, Sven %A Kamp, Martin %A Sauer, Markus %A Heinze, Katrin G. %D 2018 %J Light: Science & Applications %N 1 %P 99-- %R 10.1038/s41377-018-0104-z %T Sharpening emitter localization in front of a tuned mirror %U https://doi.org/10.1038/s41377-018-0104-z %V 7 %X Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
• The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling. Börtlein, Charlene; Draeger, Annette; Schoenauer, Roman; Kuhlemann, Alexander; Sauer, Markus; Schneider-Schaulies, Sibylle; Avota, Elita. In Frontiers in Immunology, 9, S. 815-. 2018.
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  By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.

  @article{bortlein2018neutral, abstract = {By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.}, author = {Börtlein, Charlene and Draeger, Annette and Schoenauer, Roman and Kuhlemann, Alexander and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, journal = {Frontiers in Immunology}, keywords = {sauer}, pages = {815–}, title = {The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling}, volume = 9, year = 2018 }

  %0 Journal Article %1 bortlein2018neutral %A Börtlein, Charlene %A Draeger, Annette %A Schoenauer, Roman %A Kuhlemann, Alexander %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2018 %J Frontiers in Immunology %P 815-- %T The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling %U https://www.frontiersin.org/article/10.3389/fimmu.2018.00815 %V 9 %X By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.

• Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species. Markert, Sebastian M.; Bauer, Vivien; Muenz, Thomas S.; Jones, Nicola G.; Helmprobst, Frederik; Britz, Sebastian; Sauer, Markus; Rössler, Wolfgang; Engstler, Markus; Stigloher, Christian. In Correlative Light and Electron Microscopy III, Bd. 140, T. Müller-Reichert, P. Verkade (Hrsg.), S. 21–47. Academic Press, 2017.
  • [ Abstract ]
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  Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.

  @inbook{markert2017chapter, abstract = {Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.}, author = {Markert, Sebastian M. and Bauer, Vivien and Muenz, Thomas S. and Jones, Nicola G. and Helmprobst, Frederik and Britz, Sebastian and Sauer, Markus and Rössler, Wolfgang and Engstler, Markus and Stigloher, Christian}, booktitle = {Correlative Light and Electron Microscopy III}, editor = {Müller-Reichert, Thomas and Verkade, Paul}, keywords = {sauer}, number = {Supplement C}, pages = {21–47}, publisher = {Academic Press}, title = {Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species}, volume = 140, year = 2017 }

  %0 Book Section %1 markert2017chapter %A Markert, Sebastian M. %A Bauer, Vivien %A Muenz, Thomas S. %A Jones, Nicola G. %A Helmprobst, Frederik %A Britz, Sebastian %A Sauer, Markus %A Rössler, Wolfgang %A Engstler, Markus %A Stigloher, Christian %B Correlative Light and Electron Microscopy III %D 2017 %E Müller-Reichert, Thomas %E Verkade, Paul %I Academic Press %N Supplement C %P 21--47 %R https://doi.org/10.1016/bs.mcb.2017.03.004 %T Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species %U http://www.sciencedirect.com/science/article/pii/S0091679X17300481 %V 140 %X Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.
• Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons. Scholz, Nicole; Guan, Chonglin; Nieberler, Matthias; Grotemeyer, Alexander; Maiellaro, Isabella; Gao, Shiqiang; Beck, Sebastian; Pawlak, Matthias; Sauer, Markus; Asan, Esther; Rothemund, Sven; Winkler, Jana; Prömel, Simone; Nagel, Georg; Langenhan, Tobias; Kittel, Robert J. In eLife, 6, H. J. Bellen (Hrsg.), S. e28360-. eLife Sciences Publications, Ltd, 2017.
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  Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

  @article{scholz2017mechanodependent, abstract = {Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.}, author = {Scholz, Nicole and Guan, Chonglin and Nieberler, Matthias and Grotemeyer, Alexander and Maiellaro, Isabella and Gao, Shiqiang and Beck, Sebastian and Pawlak, Matthias and Sauer, Markus and Asan, Esther and Rothemund, Sven and Winkler, Jana and Prömel, Simone and Nagel, Georg and Langenhan, Tobias and Kittel, Robert J}, editor = {Bellen, Hugo J}, journal = {eLife}, keywords = {sauer}, pages = {e28360–}, publisher = {eLife Sciences Publications, Ltd}, title = {Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons}, volume = 6, year = 2017 }

  %0 Journal Article %1 scholz2017mechanodependent %A Scholz, Nicole %A Guan, Chonglin %A Nieberler, Matthias %A Grotemeyer, Alexander %A Maiellaro, Isabella %A Gao, Shiqiang %A Beck, Sebastian %A Pawlak, Matthias %A Sauer, Markus %A Asan, Esther %A Rothemund, Sven %A Winkler, Jana %A Prömel, Simone %A Nagel, Georg %A Langenhan, Tobias %A Kittel, Robert J %D 2017 %E Bellen, Hugo J %I eLife Sciences Publications, Ltd %J eLife %P e28360-- %R 10.7554/eLife.28360 %T Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons %U https://doi.org/10.7554/eLife.28360 %V 6 %X Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
• CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells. Ziegler, Sabrina; Weiss, Esther; Schmitt, Anna-Lena; Schlegel, Jan; Burgert, Anne; Terpitz, Ulrich; Sauer, Markus; Moretta, Lorenzo; Sivori, Simona; Leonhardt, Ines; Kurzai, Oliver; Einsele, Hermann; Loeffler, Juergen. In Scientific Reports, 7(1), S. 6138-. 2017.
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  Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

  @article{ziegler2017pathogen, abstract = {Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.}, author = {Ziegler, Sabrina and Weiss, Esther and Schmitt, Anna-Lena and Schlegel, Jan and Burgert, Anne and Terpitz, Ulrich and Sauer, Markus and Moretta, Lorenzo and Sivori, Simona and Leonhardt, Ines and Kurzai, Oliver and Einsele, Hermann and Loeffler, Juergen}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {6138–}, title = {CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells}, volume = 7, year = 2017 }

  %0 Journal Article %1 ziegler2017pathogen %A Ziegler, Sabrina %A Weiss, Esther %A Schmitt, Anna-Lena %A Schlegel, Jan %A Burgert, Anne %A Terpitz, Ulrich %A Sauer, Markus %A Moretta, Lorenzo %A Sivori, Simona %A Leonhardt, Ines %A Kurzai, Oliver %A Einsele, Hermann %A Loeffler, Juergen %D 2017 %J Scientific Reports %N 1 %P 6138-- %R 10.1038/s41598-017-06238-4 %T CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells %U https://doi.org/10.1038/s41598-017-06238-4 %V 7 %X Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
• Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation. Brühlmann, David; Muhr, Anais; Parker, Rebecca; Vuillemin, Thomas; Bucsella, Blanka; Kalman, Franka; Torre, Serena; La Neve, Fabio; Lembo, Antonio; Haas, Tobias; Sauer, Markus; Souquet, Jonathan; Broly, Hervé; Hemberger, Jürgen; Jordan, Martin. In Journal of Biotechnology, 252(Supplement C), S. 32–42. 2017.
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  Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.

  @article{bruhlmann2017culture, abstract = {Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.}, author = {Brühlmann, David and Muhr, Anais and Parker, Rebecca and Vuillemin, Thomas and Bucsella, Blanka and Kalman, Franka and Torre, Serena and La Neve, Fabio and Lembo, Antonio and Haas, Tobias and Sauer, Markus and Souquet, Jonathan and Broly, Hervé and Hemberger, Jürgen and Jordan, Martin}, journal = {Journal of Biotechnology}, keywords = {sauer}, number = {Supplement C}, pages = {32–42}, title = {Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation}, volume = 252, year = 2017 }

  %0 Journal Article %1 bruhlmann2017culture %A Brühlmann, David %A Muhr, Anais %A Parker, Rebecca %A Vuillemin, Thomas %A Bucsella, Blanka %A Kalman, Franka %A Torre, Serena %A La Neve, Fabio %A Lembo, Antonio %A Haas, Tobias %A Sauer, Markus %A Souquet, Jonathan %A Broly, Hervé %A Hemberger, Jürgen %A Jordan, Martin %D 2017 %J Journal of Biotechnology %N Supplement C %P 32--42 %R https://doi.org/10.1016/j.jbiotec.2017.04.026 %T Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation %U http://www.sciencedirect.com/science/article/pii/S016816561730189X %V 252 %X Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.
• Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality. Brühlmann, David; Sokolov, Michael; Butté, Alessandro; Sauer, Markus; Hemberger, Jürgen; Souquet, Jonathan; Broly, Hervé; Jordan, Martin. In Biotechnology and Bioengineering, 114(7), S. 1448–1458. 2017.
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  Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.

  @article{bruhlmann2017parallel, abstract = {Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.}, author = {Brühlmann, David and Sokolov, Michael and Butté, Alessandro and Sauer, Markus and Hemberger, Jürgen and Souquet, Jonathan and Broly, Hervé and Jordan, Martin}, journal = {Biotechnology and Bioengineering}, keywords = {sauer}, number = 7, pages = {1448–1458}, title = {Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality}, volume = 114, year = 2017 }

  %0 Journal Article %1 bruhlmann2017parallel %A Brühlmann, David %A Sokolov, Michael %A Butté, Alessandro %A Sauer, Markus %A Hemberger, Jürgen %A Souquet, Jonathan %A Broly, Hervé %A Jordan, Martin %D 2017 %J Biotechnology and Bioengineering %N 7 %P 1448--1458 %R 10.1002/bit.26269 %T Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality %U http://dx.doi.org/10.1002/bit.26269 %V 114 %X Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.
• Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy. Burgert, Anne; Schlegel, Jan; Bécam, Jérôme; Doose, Sören; Bieberich, Erhard; Schubert-Unkmeir, Alexandra; Sauer, Markus. In Angewandte Chemie. 2017.
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  The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

  @article{burgert2017characterization, abstract = {The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.}, author = {Burgert, Anne and Schlegel, Jan and Bécam, Jérôme and Doose, Sören and Bieberich, Erhard and Schubert-Unkmeir, Alexandra and Sauer, Markus}, journal = {Angewandte Chemie}, keywords = {sauer}, title = {Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy}, year = 2017 }

  %0 Journal Article %1 burgert2017characterization %A Burgert, Anne %A Schlegel, Jan %A Bécam, Jérôme %A Doose, Sören %A Bieberich, Erhard %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2017 %J Angewandte Chemie %R 10.1002/ange.201700570 %T Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201700570 %X The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.
• Single-Molecule Localization Microscopy in Eukaryotes. Sauer, Markus; Heilemann, Mike. In Chem. Rev. American Chemical Society, 2017.
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  Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.

  @article{sauer2017singlemolecule, abstract = {Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.}, author = {Sauer, Markus and Heilemann, Mike}, booktitle = {Chemical Reviews}, journal = {Chem. Rev.}, keywords = {mike}, month = {03}, publisher = {American Chemical Society}, title = {Single-Molecule Localization Microscopy in Eukaryotes}, year = 2017 }

  %0 Journal Article %1 sauer2017singlemolecule %A Sauer, Markus %A Heilemann, Mike %B Chemical Reviews %D 2017 %I American Chemical Society %J Chem. Rev. %R 10.1021/acs.chemrev.6b00667 %T Single-Molecule Localization Microscopy in Eukaryotes %U http://dx.doi.org/10.1021/acs.chemrev.6b00667 %X Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.
• Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain. Forero, Andrea; Rivero, Olga; Wäldchen, Sina; Ku, Hsing-Ping; Kiser, Dominik P.; Gärtner, Yvonne; Pennington, Laura S.; Waider, Jonas; Gaspar, Patricia; Jansch, Charline; Edenhofer, Frank; Resink, Thérèse J.; Blum, Robert; Sauer, Markus; Lesch, Klaus-Peter. In Frontiers in Cellular Neuroscience, 11, S. 307-. 2017.
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  Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.

  @article{forero2017cadherin13, abstract = {Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.}, author = {Forero, Andrea and Rivero, Olga and Wäldchen, Sina and Ku, Hsing-Ping and Kiser, Dominik P. and Gärtner, Yvonne and Pennington, Laura S. and Waider, Jonas and Gaspar, Patricia and Jansch, Charline and Edenhofer, Frank and Resink, Thérèse J. and Blum, Robert and Sauer, Markus and Lesch, Klaus-Peter}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, pages = {307–}, title = {Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain}, volume = 11, year = 2017 }

  %0 Journal Article %1 forero2017cadherin13 %A Forero, Andrea %A Rivero, Olga %A Wäldchen, Sina %A Ku, Hsing-Ping %A Kiser, Dominik P. %A Gärtner, Yvonne %A Pennington, Laura S. %A Waider, Jonas %A Gaspar, Patricia %A Jansch, Charline %A Edenhofer, Frank %A Resink, Thérèse J. %A Blum, Robert %A Sauer, Markus %A Lesch, Klaus-Peter %D 2017 %J Frontiers in Cellular Neuroscience %P 307-- %T Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain %U https://www.frontiersin.org/article/10.3389/fncel.2017.00307 %V 11 %X Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
• Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging. Lukeš, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Levet, Florian; Benda, Aleš; Letschert, Sebastian; Sauer, Markus; Brdička, Tomáš; Lasser, Theo; Cebecauer, Marek. In Nature Communications, 8(1), S. 1731-. 2017.
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  Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.

  @article{lukes2017quantifying, abstract = {Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.}, author = {Lukeš, Tomáš and Glatzová, Daniela and Kvíčalová, Zuzana and Levet, Florian and Benda, Aleš and Letschert, Sebastian and Sauer, Markus and Brdička, Tomáš and Lasser, Theo and Cebecauer, Marek}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {1731–}, title = {Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging}, volume = 8, year = 2017 }

  %0 Journal Article %1 lukes2017quantifying %A Lukeš, Tomáš %A Glatzová, Daniela %A Kvíčalová, Zuzana %A Levet, Florian %A Benda, Aleš %A Letschert, Sebastian %A Sauer, Markus %A Brdička, Tomáš %A Lasser, Theo %A Cebecauer, Marek %D 2017 %J Nature Communications %N 1 %P 1731-- %R 10.1038/s41467-017-01857-x %T Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging %U https://doi.org/10.1038/s41467-017-01857-x %V 8 %X Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
• Incorporation studies of clickable ceramides in Jurkat cell plasma membranes. Walter, T.; Schlegel, J.; Burgert, A.; Kurz, A.; Seibel, J.; Sauer, M. In Chem. Commun., 53(51), S. 6836–6839. The Royal Society of Chemistry, 2017.
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  The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.

  @article{walter2017incorporation, abstract = {The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.}, author = {Walter, T. and Schlegel, J. and Burgert, A. and Kurz, A. and Seibel, J. and Sauer, M.}, journal = {Chem. Commun.}, keywords = {sauer}, number = 51, pages = {6836–6839}, publisher = {The Royal Society of Chemistry}, title = {Incorporation studies of clickable ceramides in Jurkat cell plasma membranes}, volume = 53, year = 2017 }

  %0 Journal Article %1 walter2017incorporation %A Walter, T. %A Schlegel, J. %A Burgert, A. %A Kurz, A. %A Seibel, J. %A Sauer, M. %D 2017 %I The Royal Society of Chemistry %J Chem. Commun. %N 51 %P 6836--6839 %R 10.1039/C7CC01220A %T Incorporation studies of clickable ceramides in Jurkat cell plasma membranes %U http://dx.doi.org/10.1039/C7CC01220A %V 53 %X The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.
• Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities. Memmel, Simon; Sisario, Dmitri; Zöller, Caren; Fiedler, Vanessa; Katzer, Astrid; Heiden, Robin; Becker, Nicholas; Eing, Lorenz; Ferreira, Fábio L.R.; Zimmermann, Heiko; Sauer, Markus; Flentje, Michael; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S. In Oncotarget, 8, S. 45298–45310. 2017.
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  High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

  @article{memmel2017migration, abstract = {High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.}, author = {Memmel, Simon and Sisario, Dmitri and Zöller, Caren and Fiedler, Vanessa and Katzer, Astrid and Heiden, Robin and Becker, Nicholas and Eing, Lorenz and Ferreira, Fábio L.R. and Zimmermann, Heiko and Sauer, Markus and Flentje, Michael and Sukhorukov, Vladimir L. and Djuzenova, Cholpon S.}, journal = {Oncotarget}, keywords = {sauer}, month = {04}, pages = {45298–45310}, title = {Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities}, volume = 8, year = 2017 }

  %0 Journal Article %1 memmel2017migration %A Memmel, Simon %A Sisario, Dmitri %A Zöller, Caren %A Fiedler, Vanessa %A Katzer, Astrid %A Heiden, Robin %A Becker, Nicholas %A Eing, Lorenz %A Ferreira, Fábio L.R. %A Zimmermann, Heiko %A Sauer, Markus %A Flentje, Michael %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %D 2017 %J Oncotarget %P 45298–45310 %T Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities %U https://doi.org/10.18632/oncotarget.16847 %V 8 %X High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
• Cyanine Conformational Restraint in the Far-Red Range. Michie, Megan S.; Götz, Ralph; Franke, Christian; Bowler, Matthew; Kumari, Nikita; Magidson, Valentin; Levitus, Marcia; Loncarek, Jadranka; Sauer, Markus; Schnermann, Martin J. In J. Am. Chem. Soc., 139(36), S. 12406–12409. American Chemical Society, 2017.
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  Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

  @article{michie2017cyanine, abstract = {Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.}, author = {Michie, Megan S. and Götz, Ralph and Franke, Christian and Bowler, Matthew and Kumari, Nikita and Magidson, Valentin and Levitus, Marcia and Loncarek, Jadranka and Sauer, Markus and Schnermann, Martin J.}, booktitle = {Journal of the American Chemical Society}, journal = {J. Am. Chem. Soc.}, keywords = {sauer}, month = {09}, number = 36, pages = {12406–12409}, publisher = {American Chemical Society}, title = {Cyanine Conformational Restraint in the Far-Red Range}, volume = 139, year = 2017 }

  %0 Journal Article %1 michie2017cyanine %A Michie, Megan S. %A Götz, Ralph %A Franke, Christian %A Bowler, Matthew %A Kumari, Nikita %A Magidson, Valentin %A Levitus, Marcia %A Loncarek, Jadranka %A Sauer, Markus %A Schnermann, Martin J. %B Journal of the American Chemical Society %D 2017 %I American Chemical Society %J J. Am. Chem. Soc. %N 36 %P 12406--12409 %R 10.1021/jacs.7b07272 %T Cyanine Conformational Restraint in the Far-Red Range %U http://dx.doi.org/10.1021/jacs.7b07272 %V 139 %X Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
• Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission. Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska; Dalby, Nils Ole; Doose, Sören; Sauer, Markus; Kneussel, Matthias; Strømgaard, Kristian. In Nature Chemical Biology, 13, S. 153-. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2017.
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  γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

  @article{maric2016gephyrinbinding, abstract = {γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.}, author = {Maric, Hans Michael and Hausrat, Torben Johann and Neubert, Franziska and Dalby, Nils Ole and Doose, Sören and Sauer, Markus and Kneussel, Matthias and Strømgaard, Kristian}, journal = {Nature Chemical Biology}, keywords = {sauer}, month = 11, pages = {153–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission}, volume = 13, year = 2017 }

  %0 Journal Article %1 maric2016gephyrinbinding %A Maric, Hans Michael %A Hausrat, Torben Johann %A Neubert, Franziska %A Dalby, Nils Ole %A Doose, Sören %A Sauer, Markus %A Kneussel, Matthias %A Strømgaard, Kristian %D 2017 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Chemical Biology %P 153-- %T Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission %U https://doi.org/10.1038/nchembio.2246 %V 13 %X γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

• Super-resolution imaging of plasma membrane proteins with click chemistry. Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Soeren; Sauer, Markus. In Frontiers in Cell and Developmental Biology, 4, S. 98-. 2016.
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  Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.

  @article{mateosgil2016superresolution, abstract = {Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.}, author = {Mateos-Gil, Pablo and Letschert, Sebastian and Doose, Soeren and Sauer, Markus}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {98–}, title = {Super-resolution imaging of plasma membrane proteins with click chemistry}, volume = 4, year = 2016 }

  %0 Journal Article %1 mateosgil2016superresolution %A Mateos-Gil, Pablo %A Letschert, Sebastian %A Doose, Soeren %A Sauer, Markus %D 2016 %J Frontiers in Cell and Developmental Biology %P 98-- %T Super-resolution imaging of plasma membrane proteins with click chemistry %U http://journal.frontiersin.org/article/10.3389/fcell.2016.00098 %V 4 %X Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.
• Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates. Alexander, Mertsch; Sebastian, Letschert; Elisabeth, Memmel; Markus, Sauer; Jürgen, Seibel. S. 347-. 2016.
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  The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.

  @misc{alexander2016synthesis, abstract = {The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.}, author = {Alexander, Mertsch and Sebastian, Letschert and Elisabeth, Memmel and Markus, Sauer and Jürgen, Seibel}, booktitle = {Zeitschrift für Naturforschung C}, keywords = {sauer}, pages = {347–}, title = {Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates}, volume = 71, year = 2016 }

  %0 Generic %1 alexander2016synthesis %A Alexander, Mertsch %A Sebastian, Letschert %A Elisabeth, Memmel %A Markus, Sauer %A Jürgen, Seibel %B Zeitschrift für Naturforschung C %D 2016 %P 347-- %R 10.1515/znc-2016-0123 %T Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates %U https://www.degruyter.com/view/j/znc.2016.71.issue-9-10/znc-2016-0123/znc-2016-0123.xml %V 71 %X The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.
• Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome. Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian. In Neurophotonics, 3(4), S. 041802–041802. 2016.
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  Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.

  @article{markert2016filling, abstract = {Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.}, author = {Markert, Sebastian Matthias and Britz, Sebastian and Proppert, Sven and Lang, Marietta and Witvliet, Daniel and Mulcahy, Ben and Sauer, Markus and Zhen, Mei and Bessereau, Jean-Louis and Stigloher, Christian}, journal = {Neurophotonics}, keywords = {sauer}, number = 4, pages = {041802–041802}, title = {Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome}, volume = 3, year = 2016 }

  %0 Journal Article %1 markert2016filling %A Markert, Sebastian Matthias %A Britz, Sebastian %A Proppert, Sven %A Lang, Marietta %A Witvliet, Daniel %A Mulcahy, Ben %A Sauer, Markus %A Zhen, Mei %A Bessereau, Jean-Louis %A Stigloher, Christian %D 2016 %J Neurophotonics %N 4 %P 041802--041802 %T Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome %U http://dx.doi.org/10.1117/1.NPh.3.4.041802 %V 3 %X Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.
• Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling. Yadav, Preeti; Selvaraj, Bhuvaneish T.; Bender, Florian L. P.; Behringer, Marcus; Moradi, Mehri; Sivadasan, Rajeeve; Dombert, Benjamin; Blum, Robert; Asan, Esther; Sauer, Markus; Julien, Jean-Pierre; Sendtner, Michael. In Acta Neuropathologica, 132(1), S. 93–110. 2016.
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  In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

  @article{yadav2016neurofilament, abstract = {In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.}, author = {Yadav, Preeti and Selvaraj, Bhuvaneish T. and Bender, Florian L. P. and Behringer, Marcus and Moradi, Mehri and Sivadasan, Rajeeve and Dombert, Benjamin and Blum, Robert and Asan, Esther and Sauer, Markus and Julien, Jean-Pierre and Sendtner, Michael}, journal = {Acta Neuropathologica}, keywords = {sauer}, number = 1, pages = {93–110}, title = {Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling}, volume = 132, year = 2016 }

  %0 Journal Article %1 yadav2016neurofilament %A Yadav, Preeti %A Selvaraj, Bhuvaneish T. %A Bender, Florian L. P. %A Behringer, Marcus %A Moradi, Mehri %A Sivadasan, Rajeeve %A Dombert, Benjamin %A Blum, Robert %A Asan, Esther %A Sauer, Markus %A Julien, Jean-Pierre %A Sendtner, Michael %D 2016 %J Acta Neuropathologica %N 1 %P 93--110 %R 10.1007/s00401-016-1564-y %T Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling %U http://dx.doi.org/10.1007/s00401-016-1564-y %V 132 %X In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
• Photometry unlocks 3D information from 2D localization microscopy data. Franke, Christian; Sauer, Markus; van de Linde, Sebastian. In Nat Meth, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
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  We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

  @article{franke2016photometry, abstract = {We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.}, author = {Franke, Christian and Sauer, Markus and van de Linde, Sebastian}, journal = {Nat Meth}, keywords = {linde}, month = 11, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Photometry unlocks 3D information from 2D localization microscopy data}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 franke2016photometry %A Franke, Christian %A Sauer, Markus %A van de Linde, Sebastian %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Photometry unlocks 3D information from 2D localization microscopy data %U http://dx.doi.org/10.1038/nmeth.4073 %V advance online publication %X We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.
• Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Niehorster, Thomas; Loschberger, Anna; Gregor, Ingo; Kramer, Benedikt; Rahn, Hans-Jurgen; Patting, Matthias; Koberling, Felix; Enderlein, Jorg; Sauer, Markus. In Nat Meth, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
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  We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

  @article{niehorster2016multitarget, abstract = {We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).}, author = {Niehorster, Thomas and Loschberger, Anna and Gregor, Ingo and Kramer, Benedikt and Rahn, Hans-Jurgen and Patting, Matthias and Koberling, Felix and Enderlein, Jorg and Sauer, Markus}, journal = {Nat Meth}, keywords = {sauer}, month = {01}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Multi-target spectrally resolved fluorescence lifetime imaging microscopy}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 niehorster2016multitarget %A Niehorster, Thomas %A Loschberger, Anna %A Gregor, Ingo %A Kramer, Benedikt %A Rahn, Hans-Jurgen %A Patting, Matthias %A Koberling, Felix %A Enderlein, Jorg %A Sauer, Markus %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Multi-target spectrally resolved fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1038/nmeth.3740 %V advance online publication %X We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).
• A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells. Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle. In The Journal of Immunology, 196(9), S. 3951–3962. 2016.
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  Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.

  @article{collenburg2016functionalized, abstract = {Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.}, author = {Collenburg, Lena and Walter, Tim and Burgert, Anne and Müller, Nora and Seibel, Jürgen and Japtok, Lukasz and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle}, journal = {The Journal of Immunology}, keywords = {sauer}, month = {05}, number = 9, pages = {3951–3962}, title = {A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells}, volume = 196, year = 2016 }

  %0 Journal Article %1 collenburg2016functionalized %A Collenburg, Lena %A Walter, Tim %A Burgert, Anne %A Müller, Nora %A Seibel, Jürgen %A Japtok, Lukasz %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %D 2016 %J The Journal of Immunology %N 9 %P 3951--3962 %T A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells %U http://www.jimmunol.org/content/196/9/3951.abstract %V 196 %X Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
• The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Dugar, Gaurav; Svensson, Sarah L.; Bischler, Thorsten; Wäldchen, Sina; Reinhardt, Richard; Sauer, Markus; Sharma, Cynthia M. In Nature Communications, 7, S. 11667-. The Author(s), 2016.
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  The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.

  @article{dugar2016csrafliw, abstract = {The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.}, author = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Wäldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.}, journal = {Nature Communications}, keywords = {sauer}, month = {05}, pages = {11667–}, publisher = {The Author(s)}, title = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni}, volume = 7, year = 2016 }

  %0 Journal Article %1 dugar2016csrafliw %A Dugar, Gaurav %A Svensson, Sarah L. %A Bischler, Thorsten %A Wäldchen, Sina %A Reinhardt, Richard %A Sauer, Markus %A Sharma, Cynthia M. %D 2016 %I The Author(s) %J Nature Communications %P 11667-- %T The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni %U http://dx.doi.org/10.1038/ncomms11667 %V 7 %X The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.
• OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors. Jung, L A; Gebhardt, A; Koelmel, W; Ade, C P; Walz, S; Kuper, J; von Eyss, B; Letschert, S; Redel, C; d’Artista, L; Biankin, A; Zender, L; Sauer, M; Wolf, E; Evan, G; Kisker, C; Eilers, M. In Oncogene, 36, S. 1911-. Macmillan Publishers Limited, part of Springer Nature., 2016.
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  MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

  @article{jung2016omomyc, abstract = {MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.}, author = {Jung, L A and Gebhardt, A and Koelmel, W and Ade, C P and Walz, S and Kuper, J and von Eyss, B and Letschert, S and Redel, C and d'Artista, L and Biankin, A and Zender, L and Sauer, M and Wolf, E and Evan, G and Kisker, C and Eilers, M}, journal = {Oncogene}, keywords = {sauer}, month = 10, pages = {1911–}, publisher = {Macmillan Publishers Limited, part of Springer Nature.}, title = {OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors}, volume = 36, year = 2016 }

  %0 Journal Article %1 jung2016omomyc %A Jung, L A %A Gebhardt, A %A Koelmel, W %A Ade, C P %A Walz, S %A Kuper, J %A von Eyss, B %A Letschert, S %A Redel, C %A d'Artista, L %A Biankin, A %A Zender, L %A Sauer, M %A Wolf, E %A Evan, G %A Kisker, C %A Eilers, M %D 2016 %I Macmillan Publishers Limited, part of Springer Nature. %J Oncogene %P 1911-- %T OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors %U http://dx.doi.org/10.1038/onc.2016.354 %V 36 %X MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.
• Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition. Werner, Christian; Pauli, Martin; Doose, Sören; Weishaupt, Andreas; Haselmann, Holger; Grünewald, Benedikt; Sauer, Markus; Heckmann, Manfred; Toyka, Klaus V.; Asan, Esther; Sommer, Claudia; Geis, Christian. In Brain, 139(2), S. 365–379. 2016.
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  See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

  @article{werner2016human, abstract = {See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.}, author = {Werner, Christian and Pauli, Martin and Doose, Sören and Weishaupt, Andreas and Haselmann, Holger and Grünewald, Benedikt and Sauer, Markus and Heckmann, Manfred and Toyka, Klaus V. and Asan, Esther and Sommer, Claudia and Geis, Christian}, journal = {Brain}, keywords = {werner}, month = {02}, number = 2, pages = {365–379}, title = {Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition}, volume = 139, year = 2016 }

  %0 Journal Article %1 werner2016human %A Werner, Christian %A Pauli, Martin %A Doose, Sören %A Weishaupt, Andreas %A Haselmann, Holger %A Grünewald, Benedikt %A Sauer, Markus %A Heckmann, Manfred %A Toyka, Klaus V. %A Asan, Esther %A Sommer, Claudia %A Geis, Christian %D 2016 %J Brain %N 2 %P 365--379 %T Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition %U http://dx.doi.org/10.1093/brain/awv324 %V 139 %X See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

• Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution. Schücker, Katharina; Holm, Thorge; Franke, Christian; Sauer, Markus; Benavente, Ricardo. In Proceedings of the National Academy of Sciences, 112(7), S. 2029–2033. 2015.
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  Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.

  @article{Schücker17022015, abstract = {Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.}, author = {Schücker, Katharina and Holm, Thorge and Franke, Christian and Sauer, Markus and Benavente, Ricardo}, journal = {Proceedings of the National Academy of Sciences}, keywords = {sauer}, number = 7, pages = {2029-2033}, title = {Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution}, volume = 112, year = 2015 }

  %0 Journal Article %1 Schücker17022015 %A Schücker, Katharina %A Holm, Thorge %A Franke, Christian %A Sauer, Markus %A Benavente, Ricardo %D 2015 %J Proceedings of the National Academy of Sciences %N 7 %P 2029-2033 %R 10.1073/pnas.1414814112 %T Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution %U http://www.pnas.org/content/112/7/2029.abstract %V 112 %X Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.
• Super-resolution microscopy of the synaptic active zone. Ehmann, Nadine; Sauer, Markus; Kittel, Robert J. In Frontiers in Cellular Neuroscience, 9(7). 2015.
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  Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.

  @article{10.3389/fncel.2015.00007, abstract = {Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.}, author = {Ehmann, Nadine and Sauer, Markus and Kittel, Robert J}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 7, title = {Super-resolution microscopy of the synaptic active zone}, volume = 9, year = 2015 }

  %0 Journal Article %1 10.3389/fncel.2015.00007 %A Ehmann, Nadine %A Sauer, Markus %A Kittel, Robert J %D 2015 %J Frontiers in Cellular Neuroscience %N 7 %R 10.3389/fncel.2015.00007 %T Super-resolution microscopy of the synaptic active zone %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00007/abstract %V 9 %X Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.
• Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy. Grimm, Jonathan B.; Klein, Teresa; Kopek, Benjamin G.; Shtengel, Gleb; Hess, Harald F.; Sauer, Markus; Lavis, Luke D. In Angewandte Chemie, S. n/a-n/a. WILEY-VCH Verlag, 2015.
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  The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.

  @article{grimm2015synthesis, abstract = {The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.}, author = {Grimm, Jonathan B. and Klein, Teresa and Kopek, Benjamin G. and Shtengel, Gleb and Hess, Harald F. and Sauer, Markus and Lavis, Luke D.}, journal = {Angewandte Chemie}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy}, year = 2015 }

  %0 Journal Article %1 grimm2015synthesis %A Grimm, Jonathan B. %A Klein, Teresa %A Kopek, Benjamin G. %A Shtengel, Gleb %A Hess, Harald F. %A Sauer, Markus %A Lavis, Luke D. %D 2015 %I WILEY-VCH Verlag %J Angewandte Chemie %P n/a--n/a %R 10.1002/ange.201509649 %T Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201509649 %X The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.
• Quantifying molecular colocalization in live cell fluorescence microscopy. Humpert, Fabian; Yahiatène, Idir; Lummer, Martina; Sauer, Markus; Huser, Thomas. In Journal of Biophotonics, 8(1-2), S. 124–132. WILEY-VCH Verlag, 2015.
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  One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

  @article{humpert2015quantifying, abstract = {One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)}, author = {Humpert, Fabian and Yahiatène, Idir and Lummer, Martina and Sauer, Markus and Huser, Thomas}, journal = {Journal of Biophotonics}, keywords = {sauer}, number = {1-2}, pages = {124–132}, publisher = {WILEY-VCH Verlag}, title = {Quantifying molecular colocalization in live cell fluorescence microscopy}, volume = 8, year = 2015 }

  %0 Journal Article %1 humpert2015quantifying %A Humpert, Fabian %A Yahiatène, Idir %A Lummer, Martina %A Sauer, Markus %A Huser, Thomas %D 2015 %I WILEY-VCH Verlag %J Journal of Biophotonics %N 1-2 %P 124--132 %R 10.1002/jbio.201300146 %T Quantifying molecular colocalization in live cell fluorescence microscopy %U http://dx.doi.org/10.1002/jbio.201300146 %V 8 %X One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)
• Tailoring recombinant protein quality by rational media design. Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé. In Biotechnology Progress, 31(3), S. 615–629. 2015.
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  Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

  @article{bruhlmann2015tailoring, abstract = {Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015}, author = {Brühlmann, David and Jordan, Martin and Hemberger, Jürgen and Sauer, Markus and Stettler, Matthieu and Broly, Hervé}, journal = {Biotechnology Progress}, keywords = {sauer}, number = 3, pages = {615–629}, title = {Tailoring recombinant protein quality by rational media design}, volume = 31, year = 2015 }

  %0 Journal Article %1 bruhlmann2015tailoring %A Brühlmann, David %A Jordan, Martin %A Hemberger, Jürgen %A Sauer, Markus %A Stettler, Matthieu %A Broly, Hervé %D 2015 %J Biotechnology Progress %N 3 %P 615--629 %R 10.1002/btpr.2089 %T Tailoring recombinant protein quality by rational media design %U http://dx.doi.org/10.1002/btpr.2089 %V 31 %X Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015
• Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation. Paul, Mila M; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J; Heckmann, Manfred. In Frontiers in Cellular Neuroscience, 9(29). 2015.
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  The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.

  @article{10.3389/fncel.2015.00029, abstract = {The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.}, author = {Paul, Mila M and Pauli, Martin and Ehmann, Nadine and Hallermann, Stefan and Sauer, Markus and Kittel, Robert J and Heckmann, Manfred}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 29, title = {Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation}, volume = 9, year = 2015 }

  %0 Journal Article %1 10.3389/fncel.2015.00029 %A Paul, Mila M %A Pauli, Martin %A Ehmann, Nadine %A Hallermann, Stefan %A Sauer, Markus %A Kittel, Robert J %A Heckmann, Manfred %D 2015 %J Frontiers in Cellular Neuroscience %N 29 %R 10.3389/fncel.2015.00029 %T Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00029/abstract %V 9 %X The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
• Artifacts in single-molecule localization microscopy. Burgert, Anne; Letschert, Sebastian; Doose, Sören; Sauer, Markus. In Histochemistry and Cell Biology, 144, S. 123–131. 2015.
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  Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

  @article{burgert2015artifacts, abstract = {Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.}, author = {Burgert, Anne and Letschert, Sebastian and Doose, Sören and Sauer, Markus}, journal = {Histochemistry and Cell Biology}, keywords = {sauer}, pages = {123-131}, series = 2, title = {Artifacts in single-molecule localization microscopy}, volume = 144, year = 2015 }

  %0 Journal Article %1 burgert2015artifacts %A Burgert, Anne %A Letschert, Sebastian %A Doose, Sören %A Sauer, Markus %B 2 %D 2015 %J Histochemistry and Cell Biology %P 123-131 %T Artifacts in single-molecule localization microscopy %U http://dx.doi.org/10.1007/s00418-015-1340-4 %V 144 %X Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.
• Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes. Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus. In Nano Lett., 15(2), S. 1374–1381. American Chemical Society, 2015.
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  Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  @article{hennig2015instant, abstract = {Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.}, author = {Hennig, Simon and van de Linde, Sebastian and Lummer, Martina and Simonis, Matthias and Huser, Thomas and Sauer, Markus}, booktitle = {Nano Letters}, journal = {Nano Lett.}, keywords = {linde}, month = {02}, number = 2, pages = {1374–1381}, publisher = {American Chemical Society}, title = {Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes}, volume = 15, year = 2015 }

  %0 Journal Article %1 hennig2015instant %A Hennig, Simon %A van de Linde, Sebastian %A Lummer, Martina %A Simonis, Matthias %A Huser, Thomas %A Sauer, Markus %B Nano Letters %D 2015 %I American Chemical Society %J Nano Lett. %N 2 %P 1374--1381 %R 10.1021/nl504660t %T Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes %U http://dx.doi.org/10.1021/nl504660t %V 15 %X Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.
• Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy. Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U.; Westerling, Katherine M.; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L. In PLoS ONE, 10(3), S. e0119990. Public Library of Science, 2015.
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  Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

  @article{10.1371/journal.pone.0119990, abstract = {Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of  3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.}, author = {Andronic, Joseph and Shirakashi, Ryo and Pickel, Simone U. and Westerling, Katherine M. and Klein, Teresa and Holm, Thorge and Sauer, Markus and Sukhorukov, Vladimir L.}, journal = {PLoS ONE}, keywords = {sauer}, month = {03}, number = 3, pages = {e0119990}, publisher = {Public Library of Science}, title = {Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy}, volume = 10, year = 2015 }

  %0 Journal Article %1 10.1371/journal.pone.0119990 %A Andronic, Joseph %A Shirakashi, Ryo %A Pickel, Simone U. %A Westerling, Katherine M. %A Klein, Teresa %A Holm, Thorge %A Sauer, Markus %A Sukhorukov, Vladimir L. %D 2015 %I Public Library of Science %J PLoS ONE %N 3 %P e0119990 %R 10.1371/journal.pone.0119990 %T Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy %U http://dx.doi.org/10.1371%2Fjournal.pone.0119990 %V 10 %X Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
• Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium. Schneider, Johannes; Klein, Teresa; Mielich-Süss, Benjamin; Koch, Gudrun; Franke, Christian; Kuipers, Oscar P.; Kovács, Ákos T.; Sauer, Markus; Lopez, Daniel. In PLoS Genet, 11(4), S. e1005140-. Public Library of Science, 2015.
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  <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>

  @article{schneider2015spatiotemporal, abstract = {<title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>}, author = {Schneider, Johannes and Klein, Teresa and Mielich-Süss, Benjamin and Koch, Gudrun and Franke, Christian and Kuipers, Oscar P. and Kovács, Ákos T. and Sauer, Markus and Lopez, Daniel}, journal = {PLoS Genet}, keywords = {sauer}, month = {04}, number = 4, pages = {e1005140–}, publisher = {Public Library of Science}, title = {Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium}, volume = 11, year = 2015 }

  %0 Journal Article %1 schneider2015spatiotemporal %A Schneider, Johannes %A Klein, Teresa %A Mielich-Süss, Benjamin %A Koch, Gudrun %A Franke, Christian %A Kuipers, Oscar P. %A Kovács, Ákos T. %A Sauer, Markus %A Lopez, Daniel %D 2015 %I Public Library of Science %J PLoS Genet %N 4 %P e1005140-- %T Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium %U http://dx.doi.org/10.1371%2Fjournal.pgen.1005140 %V 11 %X <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>
• Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns. Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus. In ACS Nano, 9(8), S. 8122–8130. American Chemical Society, 2015.
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  We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

  @article{hennig2015quantitative, abstract = {We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.}, author = {Hennig, Simon and van de Linde, Sebastian and Bergmann, Stephan and Huser, Thomas and Sauer, Markus}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {linde}, month = {08}, number = 8, pages = {8122–8130}, publisher = {American Chemical Society}, title = {Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns}, volume = 9, year = 2015 }

  %0 Journal Article %1 hennig2015quantitative %A Hennig, Simon %A van de Linde, Sebastian %A Bergmann, Stephan %A Huser, Thomas %A Sauer, Markus %B ACS Nano %D 2015 %I American Chemical Society %J ACS Nano %N 8 %P 8122--8130 %R 10.1021/acsnano.5b02220 %T Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns %U http://dx.doi.org/10.1021/acsnano.5b02220 %V 9 %X We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.
• Light-induced cell damage in live-cell super-resolution microscopy. Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In Scientific Reports, 5, S. 15348-. Nature Publishing Group, 2015.
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  Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

  @article{waldchen2015lightinduced, abstract = {Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of  1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only  50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.}, author = {Wäldchen, Sina and Lehmann, Julian and Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {Scientific Reports}, keywords = {linde}, month = {09}, pages = {15348–}, publisher = {Nature Publishing Group}, title = {Light-induced cell damage in live-cell super-resolution microscopy}, volume = 5, year = 2015 }

  %0 Journal Article %1 waldchen2015lightinduced %A Wäldchen, Sina %A Lehmann, Julian %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2015 %I Nature Publishing Group %J Scientific Reports %P 15348-- %R 10.1038/srep15348 %T Light-induced cell damage in live-cell super-resolution microscopy %U http://dx.doi.org/10.1038/srep15348 %V 5 %X Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
• A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer. von Eyss, Björn; Jaenicke, Laura A; Kortlever, Roderik M; Royla, Nadine; Wiese, Katrin E; Letschert, Sebastian; McDuffus, Leigh-Anne; Sauer, Markus; Rosenwald, Andreas; Evan, Gerard I; Kempa, Stefan; Eilers, Martin. In Cancer Cell, 28(6), S. 743–757. 2015.
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  Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

  @article{voneyss2015mycdriven, abstract = {Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.}, author = {von Eyss, Björn and Jaenicke, Laura A and Kortlever, Roderik M and Royla, Nadine and Wiese, Katrin E and Letschert, Sebastian and McDuffus, Leigh-Anne and Sauer, Markus and Rosenwald, Andreas and Evan, Gerard I and Kempa, Stefan and Eilers, Martin}, journal = {Cancer Cell}, keywords = {sauer}, month = 12, number = 6, pages = {743–757}, title = {A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer}, volume = 28, year = 2015 }

  %0 Journal Article %1 voneyss2015mycdriven %A von Eyss, Björn %A Jaenicke, Laura A %A Kortlever, Roderik M %A Royla, Nadine %A Wiese, Katrin E %A Letschert, Sebastian %A McDuffus, Leigh-Anne %A Sauer, Markus %A Rosenwald, Andreas %A Evan, Gerard I %A Kempa, Stefan %A Eilers, Martin %D 2015 %J Cancer Cell %N 6 %P 743--757 %R http://dx.doi.org/10.1016/j.ccell.2015.10.013 %T A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer %U http://www.sciencedirect.com/science/article/pii/S1535610815003906 %V 28 %X Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

• How to switch a fluorophore: from undesired blinking to controlled photoswitching. van de Linde, Sebastian; Sauer, Markus. In Chem. Soc. Rev. 2014, 43(4), S. 1076–1087. The Royal Society of Chemistry, 2014.
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  Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.

  @article{C3CS60195A, abstract = {Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.}, author = {van de Linde, Sebastian and Sauer, Markus}, journal = {Chem. Soc. Rev. 2014}, keywords = {linde}, pages = {1076-1087}, publisher = {The Royal Society of Chemistry}, title = {How to switch a fluorophore: from undesired blinking to controlled photoswitching}, volume = {43(4)}, year = 2014 }

  %0 Journal Article %1 C3CS60195A %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I The Royal Society of Chemistry %J Chem. Soc. Rev. 2014 %P 1076-1087 %R 10.1039/C3CS60195A %T How to switch a fluorophore: from undesired blinking to controlled photoswitching %U http://dx.doi.org/10.1039/C3CS60195A %V 43(4) %X Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.
• Super-Resolution Imaging of Plasma Membrane Glycans. Letschert, Sebastian; Göhler, Antonia; Franke, Christian; Bertleff-Zieschang, Nadja; Memmel, Elisabeth; Doose, Sören; Seibel, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, S. n/a-n/a. WILEY-VCH Verlag, 2014.
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  Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.

  @article{ANIE:ANIE201406045, abstract = {Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.}, author = {Letschert, Sebastian and Göhler, Antonia and Franke, Christian and Bertleff-Zieschang, Nadja and Memmel, Elisabeth and Doose, Sören and Seibel, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Super-Resolution Imaging of Plasma Membrane Glycans}, year = 2014 }

  %0 Journal Article %1 ANIE:ANIE201406045 %A Letschert, Sebastian %A Göhler, Antonia %A Franke, Christian %A Bertleff-Zieschang, Nadja %A Memmel, Elisabeth %A Doose, Sören %A Seibel, Jürgen %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %P n/a--n/a %R 10.1002/anie.201406045 %T Super-Resolution Imaging of Plasma Membrane Glycans %U http://dx.doi.org/10.1002/anie.201406045 %X Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.
• High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons. Andreska, Thomas; Aufmkolk, Sarah; Sauer, Markus; Blum, Robert. In Frontiers in Cellular Neuroscience, 8(107). 2014.
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  In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.

  @article{10.3389/fncel.2014.00107, abstract = {In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of  20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of  60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.}, author = {Andreska, Thomas and Aufmkolk, Sarah and Sauer, Markus and Blum, Robert}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 107, title = {High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons}, volume = 8, year = 2014 }

  %0 Journal Article %1 10.3389/fncel.2014.00107 %A Andreska, Thomas %A Aufmkolk, Sarah %A Sauer, Markus %A Blum, Robert %D 2014 %J Frontiers in Cellular Neuroscience %N 107 %R 10.3389/fncel.2014.00107 %T High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2014.00107/abstract %V 8 %X In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.
• A Blueprint for Cost-Efficient Localization Microscopy. Holm, Thorge; Klein, Teresa; Löschberger, Anna; Klamp, Tobias; Wiebusch, Gerd; van de Linde, Sebastian; Sauer, Markus. In ChemPhysChem, 15(4), S. 651–654. WILEY-VCH Verlag, 2014.
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  Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.

  @article{CPHC:CPHC201300739, abstract = {Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.}, author = {Holm, Thorge and Klein, Teresa and Löschberger, Anna and Klamp, Tobias and Wiebusch, Gerd and van de Linde, Sebastian and Sauer, Markus}, journal = {ChemPhysChem}, keywords = {linde}, number = 4, pages = {651–654}, publisher = {WILEY-VCH Verlag}, title = {A Blueprint for Cost-Efficient Localization Microscopy}, volume = 15, year = 2014 }

  %0 Journal Article %1 CPHC:CPHC201300739 %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Klamp, Tobias %A Wiebusch, Gerd %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J ChemPhysChem %N 4 %P 651--654 %R 10.1002/cphc.201300739 %T A Blueprint for Cost-Efficient Localization Microscopy %U http://dx.doi.org/10.1002/cphc.201300739 %V 15 %X Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.
• Timing Protein Assembly in Neurons. Sauer, Markus. In Chemistry & Biology, 21(6), S. 703–704. 2014.
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  Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.

  @article{sauer2014timing, abstract = {Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.}, author = {Sauer, Markus}, journal = {Chemistry & Biology}, keywords = {sauer}, number = 6, pages = {703–704}, title = {Timing Protein Assembly in Neurons}, volume = 21, year = 2014 }

  %0 Journal Article %1 sauer2014timing %A Sauer, Markus %D 2014 %J Chemistry & Biology %N 6 %P 703--704 %R https://doi.org/10.1016/j.chembiol.2014.06.001 %T Timing Protein Assembly in Neurons %U http://www.sciencedirect.com/science/article/pii/S1074552114001847 %V 21 %X Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.
• The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress. Mehlitz, Adrian; Karunakaran, Karthika; Herweg, Jo-Ana; Krohne, Georg; van de Linde, Sebastian; Rieck, Elke; Sauer, Markus; Rudel, Thomas. In Cellular Microbiology, S. n/a-n/a. 2014.
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  Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.

  @article{CMI:CMI12278, abstract = {Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.}, author = {Mehlitz, Adrian and Karunakaran, Karthika and Herweg, Jo-Ana and Krohne, Georg and van de Linde, Sebastian and Rieck, Elke and Sauer, Markus and Rudel, Thomas}, journal = {Cellular Microbiology}, keywords = {linde}, pages = {n/a–n/a}, title = {The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress}, year = 2014 }

  %0 Journal Article %1 CMI:CMI12278 %A Mehlitz, Adrian %A Karunakaran, Karthika %A Herweg, Jo-Ana %A Krohne, Georg %A van de Linde, Sebastian %A Rieck, Elke %A Sauer, Markus %A Rudel, Thomas %D 2014 %J Cellular Microbiology %P n/a--n/a %R 10.1111/cmi.12278 %T The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress %U http://dx.doi.org/10.1111/cmi.12278 %X Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.
• Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution. Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus. In Journal of Cell Science, 127(20), S. 4351–4355. 2014.
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  Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

  @article{Löschberger15102014, abstract = {Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.}, author = {Löschberger, Anna and Franke, Christian and Krohne, Georg and van de Linde, Sebastian and Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {linde}, number = 20, pages = {4351-4355}, title = {Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution}, volume = 127, year = 2014 }

  %0 Journal Article %1 Löschberger15102014 %A Löschberger, Anna %A Franke, Christian %A Krohne, Georg %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %J Journal of Cell Science %N 20 %P 4351-4355 %R 10.1242/jcs.156620 %T Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution %U http://jcs.biologists.org/content/127/20/4351.abstract %V 127 %X Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
• Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Löschberger, Anna; Niehörster, Thomas; Sauer, Markus. In Biotechnology Journal, S. n/a-n/a. WILEY-VCH Verlag, 2014.
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  Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

  @article{BIOT:BIOT201400026, abstract = {Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.}, author = {Löschberger, Anna and Niehörster, Thomas and Sauer, Markus}, journal = {Biotechnology Journal}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx}, year = 2014 }

  %0 Journal Article %1 BIOT:BIOT201400026 %A Löschberger, Anna %A Niehörster, Thomas %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Biotechnology Journal %P n/a--n/a %R 10.1002/biot.201400026 %T Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx %U http://dx.doi.org/10.1002/biot.201400026 %X Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.
• Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM). Whelan, Donna R.; Holm, Thorge; Sauer, Markus; Bell, Toby D. M. In Australian Journal of Chemistry, 67, S. 179–183. 2014.
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  The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

  @article{whelan2014focus, abstract = {The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.}, author = {Whelan, Donna R. and Holm, Thorge and Sauer, Markus and Bell, Toby D. M.}, journal = {Australian Journal of Chemistry}, keywords = {sauer}, pages = {179-183}, series = 2, title = {Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)}, volume = 67, year = 2014 }

  %0 Journal Article %1 whelan2014focus %A Whelan, Donna R. %A Holm, Thorge %A Sauer, Markus %A Bell, Toby D. M. %B 2 %D 2014 %J Australian Journal of Chemistry %P 179-183 %T Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM) %U http://www.publish.csiro.au/paper/CH13499 %V 67 %X The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.
• Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity. Bielopolski, Noa; Lam, Alice D.; Bar-On, Dana; Sauer, Markus; Stuenkel, Edward L.; Ashery, Uri. In Journal of Biological Chemistry, 289, S. 17087–17099. 2014.
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  Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

  @article{bielopolski2014differential, abstract = {Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.}, author = {Bielopolski, Noa and Lam, Alice D. and Bar-On, Dana and Sauer, Markus and Stuenkel, Edward L. and Ashery, Uri}, journal = {Journal of Biological Chemistry}, keywords = {sauer}, pages = {17087-17099}, series = 24, title = {Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity}, volume = 289, year = 2014 }

  %0 Journal Article %1 bielopolski2014differential %A Bielopolski, Noa %A Lam, Alice D. %A Bar-On, Dana %A Sauer, Markus %A Stuenkel, Edward L. %A Ashery, Uri %B 24 %D 2014 %J Journal of Biological Chemistry %P 17087-17099 %T Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity %U http://www.jbc.org/content/289/24/17087.abstract %V 289 %X Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.
• PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching. Sauer, Markus; Neuweiler, Hannes. In Methods Mol Biol - Fluorescence Spectroscopy and Microscopy, Bd. 1076, Y. Engelborghs, A. J. Visser (Hrsg.), S. 597–615. Humana Press, 2014.
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  Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.

  @incollection{year={2014}, abstract = {Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.}, author = {Sauer, Markus and Neuweiler, Hannes}, booktitle = {Methods Mol Biol - Fluorescence Spectroscopy and Microscopy}, editor = {Engelborghs, Yves and Visser, Antonie J.W.G.}, keywords = {hannes}, pages = {597-615}, publisher = {Humana Press}, series = {Methods in Molecular Biology}, title = {PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching}, volume = 1076, year = 2014 }

  %0 Book Section %1 year={2014} %A Sauer, Markus %A Neuweiler, Hannes %B Methods Mol Biol - Fluorescence Spectroscopy and Microscopy %D 2014 %E Engelborghs, Yves %E Visser, Antonie J.W.G. %I Humana Press %P 597-615 %R 10.1007/978-1-62703-649-8_27 %T PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching %U http://dx.doi.org/10.1007/978-1-62703-649-8_27 %V 1076 %X Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples. %@ 978-1-62703-648-1
• Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging. Proppert, Sven; Wolter, Steve; Holm, Thorge; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In Opt. Express, 22(9), S. 10304–10316. OSA, 2014.
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  In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

  @article{Proppert:14, abstract = {In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.}, author = {Proppert, Sven and Wolter, Steve and Holm, Thorge and Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {Opt. Express}, keywords = {linde}, month = {05}, number = 9, pages = {10304–10316}, publisher = {OSA}, title = {Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging}, volume = 22, year = 2014 }

  %0 Journal Article %1 Proppert:14 %A Proppert, Sven %A Wolter, Steve %A Holm, Thorge %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I OSA %J Opt. Express %N 9 %P 10304--10316 %R 10.1364/OE.22.010304 %T Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging %U http://www.opticsexpress.org/abstract.cfm?URI=oe-22-9-10304 %V 22 %X In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
• Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Ehmann, Nadine; van de Linde, Sebastian; Alon, Amit; Ljaschenko, Dmitrij; Keung, Xi Zhen; Holm, Thorge; Rings, Annika; DiAntonio, Aaron; Hallermann, Stefan; Ashery, Uri; Heckmann, Manfred; Sauer, Markus; Kittel, Robert J. In Nat Commun, 5. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2014.
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  The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

  @article{ehmann2014quantitative, abstract = {The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing  137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.}, author = {Ehmann, Nadine and van de Linde, Sebastian and Alon, Amit and Ljaschenko, Dmitrij and Keung, Xi Zhen and Holm, Thorge and Rings, Annika and DiAntonio, Aaron and Hallermann, Stefan and Ashery, Uri and Heckmann, Manfred and Sauer, Markus and Kittel, Robert J.}, journal = {Nat Commun}, keywords = {linde}, month = {08}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states}, volume = 5, year = 2014 }

  %0 Journal Article %1 ehmann2014quantitative %A Ehmann, Nadine %A van de Linde, Sebastian %A Alon, Amit %A Ljaschenko, Dmitrij %A Keung, Xi Zhen %A Holm, Thorge %A Rings, Annika %A DiAntonio, Aaron %A Hallermann, Stefan %A Ashery, Uri %A Heckmann, Manfred %A Sauer, Markus %A Kittel, Robert J. %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states %U http://dx.doi.org/10.1038/ncomms5650 %V 5 %X The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
• Eight years of single-molecule localization microscopy. Klein, Teresa; Proppert, Sven; Sauer, Markus. In Histochemistry and Cell Biology, 141(6), S. 561–575. 2014.
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  Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

  @article{klein2014eight, abstract = {Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.}, author = {Klein, Teresa and Proppert, Sven and Sauer, Markus}, journal = {Histochemistry and Cell Biology}, keywords = {sauer}, number = 6, pages = {561–575}, title = {Eight years of single-molecule localization microscopy}, volume = 141, year = 2014 }

  %0 Journal Article %1 klein2014eight %A Klein, Teresa %A Proppert, Sven %A Sauer, Markus %D 2014 %J Histochemistry and Cell Biology %N 6 %P 561--575 %R 10.1007/s00418-014-1184-3 %T Eight years of single-molecule localization microscopy %U https://doi.org/10.1007/s00418-014-1184-3 %V 141 %X Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

• Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Andronic, Joseph; Bobak, Nicole; Bittner, Stefan; Ehling, Petra; Kleinschnitz, Christoph; Herrmann, Alexander M.; Zimmermann, Heiko; Sauer, Markus; Wiendl, Heinz; Budde, Thomas; Meuth, Sven G.; Sukhorukov, Vladimir L. In Biochimica et Biophysica Acta (BBA) - Biomembranes, 1828(2), S. 699–707. 2013.
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  Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.

  @article{Andronic2013699, abstract = {Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.}, author = {Andronic, Joseph and Bobak, Nicole and Bittner, Stefan and Ehling, Petra and Kleinschnitz, Christoph and Herrmann, Alexander M. and Zimmermann, Heiko and Sauer, Markus and Wiendl, Heinz and Budde, Thomas and Meuth, Sven G. and Sukhorukov, Vladimir L.}, journal = {Biochimica et Biophysica Acta (BBA) - Biomembranes}, keywords = {sauer}, number = 2, pages = {699 - 707}, title = {Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes}, volume = 1828, year = 2013 }

  %0 Journal Article %1 Andronic2013699 %A Andronic, Joseph %A Bobak, Nicole %A Bittner, Stefan %A Ehling, Petra %A Kleinschnitz, Christoph %A Herrmann, Alexander M. %A Zimmermann, Heiko %A Sauer, Markus %A Wiendl, Heinz %A Budde, Thomas %A Meuth, Sven G. %A Sukhorukov, Vladimir L. %D 2013 %J Biochimica et Biophysica Acta (BBA) - Biomembranes %N 2 %P 699 - 707 %R 10.1016/j.bbamem.2012.09.028 %T Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes %U http://www.sciencedirect.com/science/article/pii/S0005273612003495 %V 1828 %X Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.
• Localization microscopy coming of age: from concepts to biological impact. Sauer, Markus. In Journal of Cell Science, 126(16), S. 3505–3513. 2013.
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  Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.

  @article{Sauer15082013, abstract = {Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.}, author = {Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {sauer}, number = 16, pages = {3505-3513}, title = {Localization microscopy coming of age: from concepts to biological impact}, volume = 126, year = 2013 }

  %0 Journal Article %1 Sauer15082013 %A Sauer, Markus %D 2013 %J Journal of Cell Science %N 16 %P 3505-3513 %R 10.1242/jcs.123612 %T Localization microscopy coming of age: from concepts to biological impact %U http://jcs.biologists.org/content/126/16/3505.abstract %V 126 %X Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.
• Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives. Kolesinska, Beata; Podwysocka, Dominika J.; Rueping, Magnus A.; Seebach, Dieter; Kamena, Faustin; Walde, Peter; Sauer, Markus; Windschiegl, Barbara; Meyer-Ács, Mira; Vor der Brüggen, Marc; Giehring, Sebastian. In Chemistry & Biodiversity, 10(1), S. 1–38. WILEY-VCH Verlag, 2013.
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  After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.

  @article{CBDV:CBDV201200393, abstract = {After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.}, author = {Kolesinska, Beata and Podwysocka, Dominika J. and Rueping, Magnus A. and Seebach, Dieter and Kamena, Faustin and Walde, Peter and Sauer, Markus and Windschiegl, Barbara and Meyer-Ács, Mira and Vor der Brüggen, Marc and Giehring, Sebastian}, journal = {Chemistry & Biodiversity}, keywords = {sauer}, number = 1, pages = {1–38}, publisher = {WILEY-VCH Verlag}, title = {Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives}, volume = 10, year = 2013 }

  %0 Journal Article %1 CBDV:CBDV201200393 %A Kolesinska, Beata %A Podwysocka, Dominika J. %A Rueping, Magnus A. %A Seebach, Dieter %A Kamena, Faustin %A Walde, Peter %A Sauer, Markus %A Windschiegl, Barbara %A Meyer-Ács, Mira %A Vor der Brüggen, Marc %A Giehring, Sebastian %D 2013 %I WILEY-VCH Verlag %J Chemistry & Biodiversity %N 1 %P 1--38 %R 10.1002/cbdv.201200393 %T Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives %U http://dx.doi.org/10.1002/cbdv.201200393 %V 10 %X After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.
• Investigating Cellular Structures at the Nanoscale with Organic Fluorophores. van de Linde, Sebastian; Aufmkolk, Sarah; Franke, Christian; Holm, Thorge; Klein, Teresa; Löschberger, Anna; Proppert, Sven; Wolter, Steve; Sauer, Markus. In Chemistry & Biology, 20(1), S. 8–18. 2013.
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  Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.

  @article{van de Linde20138, abstract = {Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.}, author = {van de Linde, Sebastian and Aufmkolk, Sarah and Franke, Christian and Holm, Thorge and Klein, Teresa and Löschberger, Anna and Proppert, Sven and Wolter, Steve and Sauer, Markus}, journal = {Chemistry & Biology}, keywords = {linde}, number = 1, pages = {8 - 18}, title = {Investigating Cellular Structures at the Nanoscale with Organic Fluorophores}, volume = 20, year = 2013 }

  %0 Journal Article %1 van de Linde20138 %A van de Linde, Sebastian %A Aufmkolk, Sarah %A Franke, Christian %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Proppert, Sven %A Wolter, Steve %A Sauer, Markus %D 2013 %J Chemistry & Biology %N 1 %P 8 - 18 %R 10.1016/j.chembiol.2012.11.004 %T Investigating Cellular Structures at the Nanoscale with Organic Fluorophores %U http://www.sciencedirect.com/science/article/pii/S1074552112004280 %V 20 %X Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.
• Elements of Transcriptional Machinery Are Compatible among Plants and Mammals. Wolf, Annette; Akrap, Nina; Marg, Berenice; Galliardt, Helena; Heiligentag, Martyna; Humpert, Fabian; Sauer, Markus; Kaltschmidt, Barbara; Kaltschmidt, Christian; Seidel, Thorsten. In PLOS ONE, 8(1), S. e53737-. Public Library of Science, 2013.
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  In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.

  @article{wolf2013elements, abstract = {In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.}, author = {Wolf, Annette and Akrap, Nina and Marg, Berenice and Galliardt, Helena and Heiligentag, Martyna and Humpert, Fabian and Sauer, Markus and Kaltschmidt, Barbara and Kaltschmidt, Christian and Seidel, Thorsten}, journal = {PLOS ONE}, keywords = {sauer}, month = {01}, number = 1, pages = {e53737–}, publisher = {Public Library of Science}, title = {Elements of Transcriptional Machinery Are Compatible among Plants and Mammals}, volume = 8, year = 2013 }

  %0 Journal Article %1 wolf2013elements %A Wolf, Annette %A Akrap, Nina %A Marg, Berenice %A Galliardt, Helena %A Heiligentag, Martyna %A Humpert, Fabian %A Sauer, Markus %A Kaltschmidt, Barbara %A Kaltschmidt, Christian %A Seidel, Thorsten %D 2013 %I Public Library of Science %J PLOS ONE %N 1 %P e53737-- %T Elements of Transcriptional Machinery Are Compatible among Plants and Mammals %U https://doi.org/10.1371/journal.pone.0053737 %V 8 %X In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
• A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants. Lummer, Martina; Humpert, Fabian; Wiedenlübbert, Matthias; Sauer, Markus; Schüttpelz, Mark; Staiger, Dorothee. In Molecular Plant, 6(5), S. 1518–1530. 2013.
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  Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.

  @article{lummer2013reversibly, abstract = {Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.}, author = {Lummer, Martina and Humpert, Fabian and Wiedenlübbert, Matthias and Sauer, Markus and Schüttpelz, Mark and Staiger, Dorothee}, journal = {Molecular Plant}, keywords = {sauer}, number = 5, pages = {1518–1530}, title = {A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants}, volume = 6, year = 2013 }

  %0 Journal Article %1 lummer2013reversibly %A Lummer, Martina %A Humpert, Fabian %A Wiedenlübbert, Matthias %A Sauer, Markus %A Schüttpelz, Mark %A Staiger, Dorothee %D 2013 %J Molecular Plant %N 5 %P 1518--1530 %R https://doi.org/10.1093/mp/sst040 %T A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants %U http://www.sciencedirect.com/science/article/pii/S1674205214602240 %V 6 %X Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.
• Methylene blue- and thiol-based oxygen depletion for super-resolution imaging. Schäfer, Philip; van de Linde, Sebastian; Lehmann, Julian; Sauer, Markus; Doose, Sören. In Analytical Chemistry, 85(6), S. 3393–3400. 2013.
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  Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

  @article{doi:10.1021/ac400035k, abstract = {Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.}, author = {Schäfer, Philip and van de Linde, Sebastian and Lehmann, Julian and Sauer, Markus and Doose, Sören}, journal = {Analytical Chemistry}, keywords = {linde}, month = {02}, number = 6, pages = {3393 - 3400}, title = {Methylene blue- and thiol-based oxygen depletion for super-resolution imaging}, volume = 85, year = 2013 }

  %0 Journal Article %1 doi:10.1021/ac400035k %A Schäfer, Philip %A van de Linde, Sebastian %A Lehmann, Julian %A Sauer, Markus %A Doose, Sören %D 2013 %J Analytical Chemistry %N 6 %P 3393 - 3400 %R 10.1021/ac400035k %T Methylene blue- and thiol-based oxygen depletion for super-resolution imaging %U http://pubs.acs.org/doi/abs/10.1021/ac400035k %V 85 %X Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.
• Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics. Seibel, Jürgen; König, Simone; Göhler, Antonia; Doose, Sören; Memmel, Elisabeth; Bertleff, Nadja; Sauer, Markus. In Expert Rev Proteomics, 10(1), S. 25–31. Expert Reviews, 2013.
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  Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.

  @article{seibel2013investigating, abstract = {Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.}, author = {Seibel, Jürgen and König, Simone and Göhler, Antonia and Doose, Sören and Memmel, Elisabeth and Bertleff, Nadja and Sauer, Markus}, booktitle = {Expert Review of Proteomics}, journal = {Expert Rev Proteomics}, keywords = {sauer}, month = {02}, number = 1, pages = {25–31}, publisher = {Expert Reviews}, title = {Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics}, volume = 10, year = 2013 }

  %0 Journal Article %1 seibel2013investigating %A Seibel, Jürgen %A König, Simone %A Göhler, Antonia %A Doose, Sören %A Memmel, Elisabeth %A Bertleff, Nadja %A Sauer, Markus %B Expert Review of Proteomics %D 2013 %I Expert Reviews %J Expert Rev Proteomics %N 1 %P 25--31 %R 10.1586/epr.12.72 %T Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics %U http://dx.doi.org/10.1586/epr.12.72 %V 10 %X Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.
• Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay. Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus. In Sci. Rep., 3. Macmillan Publishers Limited. All rights reserved, 2013.
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  There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

  @article{klamp2013highly, abstract = {There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of  5 pM and a linear detection range spanning three orders of magnitude.}, author = {Klamp, Tobias and Camps, Marta and Nieto, Benjamin and Guasch, Francesc and Ranasinghe, Rohan T. and Wiedemann, Jens and Petrášek, Zdeněk and Schwille, Petra and Klenerman, David and Sauer, Markus}, journal = {Sci. Rep.}, keywords = {sauer}, month = {05}, publisher = {Macmillan Publishers Limited. All rights reserved}, title = {Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay}, volume = 3, year = 2013 }

  %0 Journal Article %1 klamp2013highly %A Klamp, Tobias %A Camps, Marta %A Nieto, Benjamin %A Guasch, Francesc %A Ranasinghe, Rohan T. %A Wiedemann, Jens %A Petrášek, Zdeněk %A Schwille, Petra %A Klenerman, David %A Sauer, Markus %D 2013 %I Macmillan Publishers Limited. All rights reserved %J Sci. Rep. %T Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay %U http://dx.doi.org/10.1038/srep01852 %V 3 %X There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

• Live-Cell Super-Resolution Imaging Goes Multicolor. Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In ChemBioChem, 13(13), S. 1861–1863. WILEY-VCH Verlag, 2012.
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  New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.

  @article{CBIC:CBIC201200347, abstract = {New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.}, author = {Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {ChemBioChem}, keywords = {linde}, number = 13, pages = {1861–1863}, publisher = {WILEY-VCH Verlag}, title = {Live-Cell Super-Resolution Imaging Goes Multicolor}, volume = 13, year = 2012 }

  %0 Journal Article %1 CBIC:CBIC201200347 %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I WILEY-VCH Verlag %J ChemBioChem %N 13 %P 1861--1863 %R 10.1002/cbic.201200347 %T Live-Cell Super-Resolution Imaging Goes Multicolor %U http://dx.doi.org/10.1002/cbic.201200347 %V 13 %X New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.
• Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters. Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian; Heilemann, Mike; Nudelman, German; Nachliel, Esther; Gutman, Menachem; Sauer, Markus; Ashery, Uri. In Journal of Biological Chemistry. 2012.
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  Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

  @article{Bar-On14062012, abstract = {Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.}, author = {Bar-On, Dana and Wolter, Steve and van de Linde, Sebastian and Heilemann, Mike and Nudelman, German and Nachliel, Esther and Gutman, Menachem and Sauer, Markus and Ashery, Uri}, journal = {Journal of Biological Chemistry}, keywords = {mike}, title = {Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters}, year = 2012 }

  %0 Journal Article %1 Bar-On14062012 %A Bar-On, Dana %A Wolter, Steve %A van de Linde, Sebastian %A Heilemann, Mike %A Nudelman, German %A Nachliel, Esther %A Gutman, Menachem %A Sauer, Markus %A Ashery, Uri %D 2012 %J Journal of Biological Chemistry %R 10.1074/jbc.M112.353250 %T Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters %U http://www.jbc.org/content/early/2012/06/14/jbc.M112.353250.abstract %X Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.
• Live-Cell Super-Resolution Imaging with Synthetic Fluorophores. van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus. In Annual Review of Physical Chemistry, 63(1), S. null. 2012.
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  Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

  @article{doi:10.1146/annurev-physchem-032811-112012, abstract = {Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.}, author = {van de Linde, Sebastian and Heilemann, Mike and Sauer, Markus}, journal = {Annual Review of Physical Chemistry}, keywords = {mike}, number = 1, pages = {null}, title = {Live-Cell Super-Resolution Imaging with Synthetic Fluorophores}, volume = 63, year = 2012 }

  %0 Journal Article %1 doi:10.1146/annurev-physchem-032811-112012 %A van de Linde, Sebastian %A Heilemann, Mike %A Sauer, Markus %D 2012 %J Annual Review of Physical Chemistry %N 1 %P null %R 10.1146/annurev-physchem-032811-112012 %T Live-Cell Super-Resolution Imaging with Synthetic Fluorophores %U http://www.annualreviews.org/doi/abs/10.1146/annurev-physchem-032811-112012 %V 63 %X Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
• Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast. Lando, David; Endesfelder, Ulrike; Berger, Harald; Subramanian, Lakxmi; Dunne, Paul D.; McColl, James; Klenerman, David; Carr, Antony M.; Sauer, Markus; Allshire, Robin C.; Heilemann, Mike; Laue, Ernest D. In Open Biology, 2(7). 2012.
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  The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

  @article{LandoJuly2012, abstract = {The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.}, author = {Lando, David and Endesfelder, Ulrike and Berger, Harald and Subramanian, Lakxmi and Dunne, Paul D. and McColl, James and Klenerman, David and Carr, Antony M. and Sauer, Markus and Allshire, Robin C. and Heilemann, Mike and Laue, Ernest D.}, journal = {Open Biology}, keywords = {mike}, number = 7, title = {Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast}, volume = 2, year = 2012 }

  %0 Journal Article %1 LandoJuly2012 %A Lando, David %A Endesfelder, Ulrike %A Berger, Harald %A Subramanian, Lakxmi %A Dunne, Paul D. %A McColl, James %A Klenerman, David %A Carr, Antony M. %A Sauer, Markus %A Allshire, Robin C. %A Heilemann, Mike %A Laue, Ernest D. %D 2012 %J Open Biology %N 7 %R 10.1098/rsob.120078 %T Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast %U http://rsob.royalsocietypublishing.org/content/2/7/120078.abstract %V 2 %X The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
• Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. Löschberger, Anna; van de Linde, Sebastian; Dabauvalle, Marie-Christine; Rieger, Bernd; Heilemann, Mike; Krohne, Georg; Sauer, Markus. In Journal of Cell Science, 125(3), S. 570–575. 2012.
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  One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

  @article{Löschberger01022012, abstract = {One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of  15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.}, author = {Löschberger, Anna and van de Linde, Sebastian and Dabauvalle, Marie-Christine and Rieger, Bernd and Heilemann, Mike and Krohne, Georg and Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {mike}, number = 3, pages = {570-575}, title = {Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution}, volume = 125, year = 2012 }

  %0 Journal Article %1 Löschberger01022012 %A Löschberger, Anna %A van de Linde, Sebastian %A Dabauvalle, Marie-Christine %A Rieger, Bernd %A Heilemann, Mike %A Krohne, Georg %A Sauer, Markus %D 2012 %J Journal of Cell Science %N 3 %P 570-575 %R 10.1242/jcs.098822 %T Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution %U http://jcs.biologists.org/content/125/3/570.abstract %V 125 %X One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.
• rapidSTORM: accurate, fast open-source software for localization microscopy. Wolter, Steve; Loschberger, Anna; Holm, Thorge; Aufmkolk, Sarah; Dabauvalle, Marie-Christine; van de Linde, Sebastian; Sauer, Markus. In Nat Meth, 9(11), S. 1040–1041. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2012.
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  To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.

  @article{wolter2012rapidstorm, abstract = {To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.}, author = {Wolter, Steve and Loschberger, Anna and Holm, Thorge and Aufmkolk, Sarah and Dabauvalle, Marie-Christine and van de Linde, Sebastian and Sauer, Markus}, journal = {Nat Meth}, keywords = {linde}, month = 11, number = 11, pages = {1040–1041}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {rapidSTORM: accurate, fast open-source software for localization microscopy}, volume = 9, year = 2012 }

  %0 Journal Article %1 wolter2012rapidstorm %A Wolter, Steve %A Loschberger, Anna %A Holm, Thorge %A Aufmkolk, Sarah %A Dabauvalle, Marie-Christine %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %N 11 %P 1040--1041 %T rapidSTORM: accurate, fast open-source software for localization microscopy %U http://dx.doi.org/10.1038/nmeth.2224 %V 9 %X To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.
• Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images. WOLTER, S.; SAUER, M. In Journal of Microscopy, 245(1), S. 109–109. Blackwell Publishing Ltd, 2012.
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  @article{JMI:JMI3562, author = {WOLTER, S. and SAUER, M.}, journal = {Journal of Microscopy}, keywords = {mike}, number = 1, pages = {109–109}, publisher = {Blackwell Publishing Ltd}, title = {Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images}, volume = 245, year = 2012 }

  %0 Journal Article %1 JMI:JMI3562 %A WOLTER, S. %A SAUER, M. %D 2012 %I Blackwell Publishing Ltd %J Journal of Microscopy %N 1 %P 109--109 %R 10.1111/j.1365-2818.2011.03562.x %T Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images %U http://dx.doi.org/10.1111/j.1365-2818.2011.03562.x %V 245
• Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments. Yahiatène, Idir; Doose, Sören; Huser, Thomas; Sauer, Markus. In Analytical Chemistry, 84(6), S. 2729–2736. 2012.
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  We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.

  @article{doi:10.1021/ac2030283, abstract = {We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.}, author = {Yahiatène, Idir and Doose, Sören and Huser, Thomas and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 6, pages = {2729-2736}, title = {Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments}, volume = 84, year = 2012 }

  %0 Journal Article %1 doi:10.1021/ac2030283 %A Yahiatène, Idir %A Doose, Sören %A Huser, Thomas %A Sauer, Markus %D 2012 %J Analytical Chemistry %N 6 %P 2729-2736 %R 10.1021/ac2030283 %T Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments %U http://pubs.acs.org/doi/abs/10.1021/ac2030283 %V 84 %X We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.
• Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies. Terpitz, Ulrich; Letschert, Sebastian; Bonda, Ulrich; Spahn, Christoph; Guan, Chonglin; Sauer, Markus; Zimmermann, Ulrich; Bamberg, Ernst; Zimmermann, Dirk; Sukhorukov, Vladimir. In Journal of Membrane Biology, S. 1–12. Springer New York, 2012.
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  The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

  @article{springerlink:10.1007/s00232-012-9484-9, abstract = {The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.}, author = {Terpitz, Ulrich and Letschert, Sebastian and Bonda, Ulrich and Spahn, Christoph and Guan, Chonglin and Sauer, Markus and Zimmermann, Ulrich and Bamberg, Ernst and Zimmermann, Dirk and Sukhorukov, Vladimir}, journal = {Journal of Membrane Biology}, keywords = {terpitz}, note = {10.1007/s00232-012-9484-9}, pages = {1-12}, publisher = {Springer New York}, title = {Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies}, year = 2012 }

  %0 Journal Article %1 springerlink:10.1007/s00232-012-9484-9 %A Terpitz, Ulrich %A Letschert, Sebastian %A Bonda, Ulrich %A Spahn, Christoph %A Guan, Chonglin %A Sauer, Markus %A Zimmermann, Ulrich %A Bamberg, Ernst %A Zimmermann, Dirk %A Sukhorukov, Vladimir %D 2012 %I Springer New York %J Journal of Membrane Biology %P 1-12 %T Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies %U http://dx.doi.org/10.1007/s00232-012-9484-9 %X The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
• Superresolution Optical Fluctuation Imaging (SOFI). Dertinger, Thomas; Colyer, Ryan; Vogel, Robert; Heilemann, Mike; Sauer, Markus; Enderlein, Jörg; Weiss, Shimon. In Advances in Experimental Medicine and Biology, 733, E. Zahavy; A. Ordentlich; S. Yitzhaki; A. Shafferman (Hrsg.), S. 17–21. Springer Netherlands, 2012.
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  Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.

  @article{springerlink:10.1007/978-94-007-2555-3_2, abstract = {Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.}, author = {Dertinger, Thomas and Colyer, Ryan and Vogel, Robert and Heilemann, Mike and Sauer, Markus and Enderlein, Jörg and Weiss, Shimon}, booktitle = {Nano-Biotechnology for Biomedical and Diagnostic Research}, editor = {Zahavy, Eran and Ordentlich, Arie and Yitzhaki, Shmuel and Shafferman, Avigdor}, journal = {Advances in Experimental Medicine and Biology}, keywords = {mike}, note = {10.1007/978-94-007-2555-3_2}, pages = {17-21}, publisher = {Springer Netherlands}, series = {Advances in Experimental Medicine and Biology}, title = {Superresolution Optical Fluctuation Imaging (SOFI)}, volume = 733, year = 2012 }

  %0 Journal Article %1 springerlink:10.1007/978-94-007-2555-3_2 %A Dertinger, Thomas %A Colyer, Ryan %A Vogel, Robert %A Heilemann, Mike %A Sauer, Markus %A Enderlein, Jörg %A Weiss, Shimon %B Nano-Biotechnology for Biomedical and Diagnostic Research %D 2012 %E Zahavy, Eran %E Ordentlich, Arie %E Yitzhaki, Shmuel %E Shafferman, Avigdor %I Springer Netherlands %J Advances in Experimental Medicine and Biology %P 17-21 %T Superresolution Optical Fluctuation Imaging (SOFI) %U http://dx.doi.org/10.1007/978-94-007-2555-3_2 %V 733 %X Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances. %@ 978-94-007-2555-3
• Fluorescence localization microscopy: The transition from concept to biological research tool. Owen, Dylan M.; Sauer, Markus; Gaus, Katharina. In cib, 5(1942-0889), S. 0–1. Landes Bioscience Inc., 2012.
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  Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.

  @article{owen2012fluorescence, abstract = {Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.}, author = {Owen, Dylan M. and Sauer, Markus and Gaus, Katharina}, journal = {cib}, keywords = {sauer}, month = {07}, number = {1942-0889}, pages = {0—1}, publisher = {Landes Bioscience Inc.}, title = {Fluorescence localization microscopy: The transition from concept to biological research tool}, volume = 5, year = 2012 }

  %0 Journal Article %1 owen2012fluorescence %A Owen, Dylan M. %A Sauer, Markus %A Gaus, Katharina %D 2012 %I Landes Bioscience Inc. %J cib %N 1942-0889 %P 0---1 %T Fluorescence localization microscopy: The transition from concept to biological research tool %U http://www.landesbioscience.com/journals/cib/article/20348/ %V 5 %X Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.

• Measuring localization performance of super-resolution algorithms on very active samples. Wolter, Steve; Endesfelder, Ulrike; van de Linde, S.; Heilemann, Mike; Sauer, Markus. In Optics Express, 19(8), S. 7020–33. 2011.
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  Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.

  @article{Sauer_2011, abstract = {Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.}, author = {Wolter, Steve and Endesfelder, Ulrike and van de Linde, S. and Heilemann, Mike and Sauer, Markus}, journal = {Optics Express}, keywords = {mike}, number = 8, pages = {7020–33}, title = {Measuring localization performance of super-resolution algorithms on very active samples.}, volume = 19, year = 2011 }

  %0 Journal Article %1 Sauer_2011 %A Wolter, Steve %A Endesfelder, Ulrike %A van de Linde, S. %A Heilemann, Mike %A Sauer, Markus %D 2011 %J Optics Express %N 8 %P 7020--33 %T Measuring localization performance of super-resolution algorithms on very active samples. %U http://www.ncbi.nlm.nih.gov/pubmed/21503016 %V 19 %X Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.
• Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Lummer, Martina; Humpert, Fabian; Steuwe, Christian; Caesar, Katharina; Schüttpelz, Mark; Sauer, Markus; Staiger, Dorothee. In Traffic, 12(6), S. 693–702. Blackwell Publishing Ltd, 2011.
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  Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.

  @article{lummer2011reversible, abstract = {Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.}, author = {Lummer, Martina and Humpert, Fabian and Steuwe, Christian and Caesar, Katharina and Schüttpelz, Mark and Sauer, Markus and Staiger, Dorothee}, journal = {Traffic}, keywords = {sauer}, number = 6, pages = {693–702}, publisher = {Blackwell Publishing Ltd}, title = {Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants}, volume = 12, year = 2011 }

  %0 Journal Article %1 lummer2011reversible %A Lummer, Martina %A Humpert, Fabian %A Steuwe, Christian %A Caesar, Katharina %A Schüttpelz, Mark %A Sauer, Markus %A Staiger, Dorothee %D 2011 %I Blackwell Publishing Ltd %J Traffic %N 6 %P 693--702 %R 10.1111/j.1600-0854.2011.01180.x %T Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants %U http://dx.doi.org/10.1111/j.1600-0854.2011.01180.x %V 12 %X Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.
• Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments. Noe, Frank; Doose, Sören; Daidone, Isabella; Löllmann, Marc; Sauer, Markus; Chodera, John D.; Smith, Jeremy C. In Proceedings of the National Academy of Sciences. 2011.
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  There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprints� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.

  @article{Noe, abstract = {There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprintsâ€� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.}, author = {Noe, Frank and Doose, Sören and Daidone, Isabella and Löllmann, Marc and Sauer, Markus and Chodera, John D. and Smith, Jeremy C.}, journal = {Proceedings of the National Academy of Sciences}, keywords = {sauer}, title = {Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments}, year = 2011 }

  %0 Journal Article %1 Noe %A Noe, Frank %A Doose, Sören %A Daidone, Isabella %A Löllmann, Marc %A Sauer, Markus %A Chodera, John D. %A Smith, Jeremy C. %D 2011 %J Proceedings of the National Academy of Sciences %T Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments %U http://dx.doi.org/10.1073/pnas.1004646108 %X There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprintsâ€� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.
• Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes. Seefeldt, Britta; Altenhoner, Kai; Tosic, Oliver; Geisler, Thomas; Sauer, Markus; Mattay, Jochen. In Photochem. Photobiol. Sci., 10(9), S. 1488–1495. The Royal Society of Chemistry, 2011.
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  We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.

  @article{seefeldt2011kinetic, abstract = {We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.}, author = {Seefeldt, Britta and Altenhoner, Kai and Tosic, Oliver and Geisler, Thomas and Sauer, Markus and Mattay, Jochen}, journal = {Photochem. Photobiol. Sci.}, keywords = {sauer}, number = 9, pages = {1488–1495}, publisher = {The Royal Society of Chemistry}, title = {Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes}, volume = 10, year = 2011 }

  %0 Journal Article %1 seefeldt2011kinetic %A Seefeldt, Britta %A Altenhoner, Kai %A Tosic, Oliver %A Geisler, Thomas %A Sauer, Markus %A Mattay, Jochen %D 2011 %I The Royal Society of Chemistry %J Photochem. Photobiol. Sci. %N 9 %P 1488--1495 %R 10.1039/C1PP05051F %T Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes %U http://dx.doi.org/10.1039/C1PP05051F %V 10 %X We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.
• Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. van de Linde, Sebastian; Krstic, Ivan; Prisner, Thomas; Doose, Soren; Heilemann, Mike; Sauer, Markus. In Photochem. Photobiol. Sci., 10(4), S. 499–506. The Royal Society of Chemistry, 2011.
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  Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.

  @article{C0PP00317D, abstract = {Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.}, author = {van de Linde, Sebastian and Krstic, Ivan and Prisner, Thomas and Doose, Soren and Heilemann, Mike and Sauer, Markus}, journal = {Photochem. Photobiol. Sci.}, keywords = {mike}, pages = {499-506}, publisher = {The Royal Society of Chemistry}, title = {Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging}, volume = 10, year = 2011 }

  %0 Journal Article %1 C0PP00317D %A van de Linde, Sebastian %A Krstic, Ivan %A Prisner, Thomas %A Doose, Soren %A Heilemann, Mike %A Sauer, Markus %D 2011 %I The Royal Society of Chemistry %J Photochem. Photobiol. Sci. %P 499-506 %R 10.1039/C0PP00317D %T Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging %U http://dx.doi.org/10.1039/C0PP00317D %V 10 %X Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.
• Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions. Bollmann, Stefan; Lollmann, Marc; Sauer, Markus; Doose, Soren. In Phys. Chem. Chem. Phys., 13(28), S. 12874–12882. The Royal Society of Chemistry, 2011.
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  Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.

  @article{C1CP21111K, abstract = {Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.}, author = {Bollmann, Stefan and Lollmann, Marc and Sauer, Markus and Doose, Soren}, journal = {Phys. Chem. Chem. Phys.}, keywords = {sauer}, pages = {12874-12882}, publisher = {The Royal Society of Chemistry}, title = {Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions}, volume = 13, year = 2011 }

  %0 Journal Article %1 C1CP21111K %A Bollmann, Stefan %A Lollmann, Marc %A Sauer, Markus %A Doose, Soren %D 2011 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %P 12874-12882 %R 10.1039/C1CP21111K %T Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions %U http://dx.doi.org/10.1039/C1CP21111K %V 13 %X Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.
• In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging. Kaminski Schierle, Gabriele S.; van de Linde, Sebastian; Erdelyi, Miklos; Esbjörner, Elin K.; Klein, Teresa; Rees, Eric; Bertoncini, Carlos W.; Dobson, Christopher M.; Sauer, Markus; Kaminski, Clemens F. In Journal of the American Chemical Society, 133(33), S. 12902–12905. 2011.
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  Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.

  @article{doi:10.1021/ja201651w, abstract = {Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.}, author = {Kaminski Schierle, Gabriele S. and van de Linde, Sebastian and Erdelyi, Miklos and Esbjörner, Elin K. and Klein, Teresa and Rees, Eric and Bertoncini, Carlos W. and Dobson, Christopher M. and Sauer, Markus and Kaminski, Clemens F.}, journal = {Journal of the American Chemical Society}, keywords = {linde}, note = {PMID: 21793568}, number = 33, pages = {12902-12905}, title = {In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging}, volume = 133, year = 2011 }

  %0 Journal Article %1 doi:10.1021/ja201651w %A Kaminski Schierle, Gabriele S. %A van de Linde, Sebastian %A Erdelyi, Miklos %A Esbjörner, Elin K. %A Klein, Teresa %A Rees, Eric %A Bertoncini, Carlos W. %A Dobson, Christopher M. %A Sauer, Markus %A Kaminski, Clemens F. %D 2011 %J Journal of the American Chemical Society %N 33 %P 12902-12905 %R 10.1021/ja201651w %T In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging %U /brokenurl# http://dx.doi.org/10.1021/ja201651w %V 133 %X Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.
• Cross-linking of DNA through HMGA1 suggests a DNA scaffold. Vogel, Benjamin; Löschberger, Anna; Sauer, Markus; Hock, Robert. In Nucleic Acids Research. 2011.
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  Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.

  @article{Vogel19052011, abstract = {Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.}, author = {Vogel, Benjamin and Löschberger, Anna and Sauer, Markus and Hock, Robert}, journal = {Nucleic Acids Research}, keywords = {sauer}, title = {Cross-linking of DNA through HMGA1 suggests a DNA scaffold}, year = 2011 }

  %0 Journal Article %1 Vogel19052011 %A Vogel, Benjamin %A Löschberger, Anna %A Sauer, Markus %A Hock, Robert %D 2011 %J Nucleic Acids Research %R 10.1093/nar/gkr396 %T Cross-linking of DNA through HMGA1 suggests a DNA scaffold %U http://nar.oxfordjournals.org/content/early/2011/05/19/nar.gkr396.abstract %X Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
• Live-cell dSTORM with SNAP-tag fusion proteins. Klein, Teresa; Loeschberger, Anna; Proppert, Sven; Wolter, Steve; van de Linde, Sebastian; Sauer, Markus. In NATURE METHODS, 8(1), S. 7–9. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2011.
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  In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.

  @article{Klein2011, abstract = {In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Klein, Teresa and Loeschberger, Anna and Proppert, Sven and Wolter, Steve and van de Linde, Sebastian and Sauer, Markus}, journal = {NATURE METHODS}, keywords = {linde}, month = {01}, number = 1, pages = {7-9}, publisher = {NATURE PUBLISHING GROUP}, title = {Live-cell dSTORM with SNAP-tag fusion proteins}, type = {Letter}, volume = 8, year = 2011 }

  %0 Journal Article %1 Klein2011 %A Klein, Teresa %A Loeschberger, Anna %A Proppert, Sven %A Wolter, Steve %A van de Linde, Sebastian %A Sauer, Markus %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2011 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 1 %P 7-9 %T Live-cell dSTORM with SNAP-tag fusion proteins %U http://dx.doi.org/10.1038/nmeth0111-7b %V 8 %X In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.
• Single-molecule Photoswitching and Localization. van de Linde, Sebastian; Wolter, Steve; Sauer, Markus. In Aust. J. Chem., 64(5), S. 503–511. 2011.
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  Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

  @article{sebastian2011singlemolecule, abstract = {Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to  20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.}, author = {van de Linde, Sebastian and Wolter, Steve and Sauer, Markus}, journal = {Aust. J. Chem.}, keywords = {linde}, month = {05}, number = 5, pages = {503–511}, title = {Single-molecule Photoswitching and Localization}, volume = 64, year = 2011 }

  %0 Journal Article %1 sebastian2011singlemolecule %A van de Linde, Sebastian %A Wolter, Steve %A Sauer, Markus %D 2011 %J Aust. J. Chem. %N 5 %P 503--511 %T Single-molecule Photoswitching and Localization %U http://dx.doi.org/10.1071/CH10284 %V 64 %X Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.
• Direct stochastic optical reconstruction microscopy with standard fluorescent probes. van de Linde, Sebastian; Loschberger, Anna; Klein, Teresa; Heidbreder, Meike; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In Nat. Protocols, 6(7), S. 991–1009. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2011.
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  Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

  @article{sebastian2011direct, abstract = {Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of  20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.}, author = {van de Linde, Sebastian and Loschberger, Anna and Klein, Teresa and Heidbreder, Meike and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {Nat. Protocols}, keywords = {mike}, month = {06}, number = 7, pages = {991–1009}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Direct stochastic optical reconstruction microscopy with standard fluorescent probes}, volume = 6, year = 2011 }

  %0 Journal Article %1 sebastian2011direct %A van de Linde, Sebastian %A Loschberger, Anna %A Klein, Teresa %A Heidbreder, Meike %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %D 2011 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat. Protocols %N 7 %P 991--1009 %T Direct stochastic optical reconstruction microscopy with standard fluorescent probes %U http://dx.doi.org/10.1038/nprot.2011.336 %V 6 %X Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.
• Conformational Flexibility of Glycosylated Peptides. Bollmann, Stefan; Burgert, Anne; Plattner, Carolin; Nagel, Lilly; Sewald, Norbert; Löllmann, Marc; Sauer, Markus; Doose, Sören. In ChemPhysChem, 12(16), S. 2907–2911. WILEY-VCH Verlag, 2011.
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  With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides

  @article{CPHC:CPHC201100650, abstract = {With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides}, author = {Bollmann, Stefan and Burgert, Anne and Plattner, Carolin and Nagel, Lilly and Sewald, Norbert and Löllmann, Marc and Sauer, Markus and Doose, Sören}, journal = {ChemPhysChem}, keywords = {sauer}, number = 16, pages = {2907–2911}, publisher = {WILEY-VCH Verlag}, title = {Conformational Flexibility of Glycosylated Peptides}, volume = 12, year = 2011 }

  %0 Journal Article %1 CPHC:CPHC201100650 %A Bollmann, Stefan %A Burgert, Anne %A Plattner, Carolin %A Nagel, Lilly %A Sewald, Norbert %A Löllmann, Marc %A Sauer, Markus %A Doose, Sören %D 2011 %I WILEY-VCH Verlag %J ChemPhysChem %N 16 %P 2907--2911 %R 10.1002/cphc.201100650 %T Conformational Flexibility of Glycosylated Peptides %U http://dx.doi.org/10.1002/cphc.201100650 %V 12 %X With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides

• Real-time computation of subdiffraction-resolution fluorescence images. Wolter, S.; Schuettpelz, M.; Tscherepanow, M.; van de Linde, S.; Heilemann, M.; Sauer, M. In JOURNAL OF MICROSCOPY-OXFORD, 237(1), S. 12–22. WILEY-BLACKWELL PUBLISHING, INC, COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA, 2010.
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  P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.

  @article{Wolter2010, abstract = {P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.}, address = {COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA}, author = {Wolter, S. and Schuettpelz, M. and Tscherepanow, M. and van de Linde, S. and Heilemann, M. and Sauer, M.}, journal = {JOURNAL OF MICROSCOPY-OXFORD}, keywords = {rapidstorm}, month = {01}, number = 1, pages = {12-22}, publisher = {WILEY-BLACKWELL PUBLISHING, INC}, title = {Real-time computation of subdiffraction-resolution fluorescence images}, type = {Article}, volume = 237, year = 2010 }

  %0 Journal Article %1 Wolter2010 %A Wolter, S. %A Schuettpelz, M. %A Tscherepanow, M. %A van de Linde, S. %A Heilemann, M. %A Sauer, M. %C COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA %D 2010 %I WILEY-BLACKWELL PUBLISHING, INC %J JOURNAL OF MICROSCOPY-OXFORD %N 1 %P 12-22 %T Real-time computation of subdiffraction-resolution fluorescence images %U http://dx.doi.org/10.1111/j.1365-2818.2009.03287.x %V 237 %X P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.
• Superresolution Optical Fluctuation Imaging with Organic Dyes. Dertinger, Thomas; Heilemann, Mike; Vogel, Robert; Sauer, Markus; Weiss, Shimon. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 49(49), S. 9441–9443. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.

  @article{Dertinger2010, abstract = {SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Dertinger, Thomas and Heilemann, Mike and Vogel, Robert and Sauer, Markus and Weiss, Shimon}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 49, pages = {9441-9443}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Superresolution Optical Fluctuation Imaging with Organic Dyes}, type = {Article}, volume = 49, year = 2010 }

  %0 Journal Article %1 Dertinger2010 %A Dertinger, Thomas %A Heilemann, Mike %A Vogel, Robert %A Sauer, Markus %A Weiss, Shimon %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 49 %P 9441-9443 %T Superresolution Optical Fluctuation Imaging with Organic Dyes %U http://dx.doi.org/10.1002/anie.201004138 %V 49 %X SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.
• Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions. Zheng, Zhikun; Nottbohm, Christoph T.; Turchanin, Andrey; Muzik, Heiko; Beyer, Andre; Heilemann, Mike; Sauer, Markus; Goelzhaeuser, Armin. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 49(45), S. 8493–8497. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.

  @article{Zheng2010, abstract = {Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Zheng, Zhikun and Nottbohm, Christoph T. and Turchanin, Andrey and Muzik, Heiko and Beyer, Andre and Heilemann, Mike and Sauer, Markus and Goelzhaeuser, Armin}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 45, pages = {8493-8497}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions}, type = {Article}, volume = 49, year = 2010 }

  %0 Journal Article %1 Zheng2010 %A Zheng, Zhikun %A Nottbohm, Christoph T. %A Turchanin, Andrey %A Muzik, Heiko %A Beyer, Andre %A Heilemann, Mike %A Sauer, Markus %A Goelzhaeuser, Armin %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 45 %P 8493-8497 %T Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions %U http://dx.doi.org/10.1002/anie.201004053 %V 49 %X Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.
• Spiropyrans as molecular optical switches. Seefeldt, Britta; Kasper, Robert; Beining, Mirco; Mattay, Jochen; Arden-Jacob, Jutta; Kemnitzer, Norbert; Drexhage, Karl Heinz; Heilemann, Mike; Sauer, Markus. In PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 9(2), S. 213–220. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2010.
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  Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.

  @article{Seefeldt2010, abstract = {Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {Seefeldt, Britta and Kasper, Robert and Beining, Mirco and Mattay, Jochen and Arden-Jacob, Jutta and Kemnitzer, Norbert and Drexhage, Karl Heinz and Heilemann, Mike and Sauer, Markus}, journal = {PHOTOCHEMICAL \& PHOTOBIOLOGICAL SCIENCES}, keywords = {mike}, number = 2, pages = {213-220}, publisher = {ROYAL SOC CHEMISTRY}, title = {Spiropyrans as molecular optical switches}, type = {Article}, volume = 9, year = 2010 }

  %0 Journal Article %1 Seefeldt2010 %A Seefeldt, Britta %A Kasper, Robert %A Beining, Mirco %A Mattay, Jochen %A Arden-Jacob, Jutta %A Kemnitzer, Norbert %A Drexhage, Karl Heinz %A Heilemann, Mike %A Sauer, Markus %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2010 %I ROYAL SOC CHEMISTRY %J PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES %N 2 %P 213-220 %T Spiropyrans as molecular optical switches %U http://dx.doi.org/10.1039/b9pp00118b %V 9 %X Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.
• Single-Molecule STED Microscopy with Photostable Organic Fluorophores. Kasper, Robert; Harke, Benjamin; Forthmann, Carsten; Tinnefeld, Philip; Hell, Stefan W.; Sauer, Markus. In Small, 6(13), T. combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing; oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic S. imaging; resolution enhancement at the single- molecule level. A. lateral resolution of (Hrsg.), S. 1379–1384. WILEY-VCH Verlag, 2010.
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  The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer

  @article{SMLL:SMLL201000203, abstract = {The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer}, author = {Kasper, Robert and Harke, Benjamin and Forthmann, Carsten and Tinnefeld, Philip and Hell, Stefan W. and Sauer, Markus}, editor = {combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing, The and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer}, journal = {Small}, keywords = {sauer}, number = 13, pages = {1379–1384}, publisher = {WILEY-VCH Verlag}, title = {Single-Molecule STED Microscopy with Photostable Organic Fluorophores}, volume = 6, year = 2010 }

  %0 Journal Article %1 SMLL:SMLL201000203 %A Kasper, Robert %A Harke, Benjamin %A Forthmann, Carsten %A Tinnefeld, Philip %A Hell, Stefan W. %A Sauer, Markus %D 2010 %E combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing, The %E imaging, oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED %E of, resolution enhancement at the single-molecule level. A lateral resolution %I WILEY-VCH Verlag %J Small %N 13 %P 1379--1384 %R 10.1002/smll.201000203 %T Single-Molecule STED Microscopy with Photostable Organic Fluorophores %U http://dx.doi.org/10.1002/smll.201000203 %V 6 %X The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer
• Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains. Daidone, Isabella; Neuweiler, Hannes; Doose, Sören; Sauer, Markus; Smith, Jeremy C. In PLOS COMPUTATIONAL BIOLOGY, 6(1). PUBLIC LIBRARY SCIENCE, 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA, 2010.
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  Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.

  @article{Daidone2010, abstract = {Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.}, address = {185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA}, author = {Daidone, Isabella and Neuweiler, Hannes and Doose, Sören and Sauer, Markus and Smith, Jeremy C.}, journal = {PLOS COMPUTATIONAL BIOLOGY}, keywords = {hannes}, month = {01}, number = 1, publisher = {PUBLIC LIBRARY SCIENCE}, title = {Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains}, type = {Article}, volume = 6, year = 2010 }

  %0 Journal Article %1 Daidone2010 %A Daidone, Isabella %A Neuweiler, Hannes %A Doose, Sören %A Sauer, Markus %A Smith, Jeremy C. %C 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA %D 2010 %I PUBLIC LIBRARY SCIENCE %J PLOS COMPUTATIONAL BIOLOGY %N 1 %T Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains %U http://dx.doi.org/10.1371/journal.pcbi.1000645 %V 6 %X Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.
• Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy. Kottke, Tilman; van de Linde, Sebastian; Sauer, Markus; Kakorin, Sergej; Heilemann, Mike. In The Journal of Physical Chemistry Letters, 1(21), S. 3156–3159. 2010.
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  The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.

  @article{doi:10.1021/jz101300t, abstract = {The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.}, author = {Kottke, Tilman and van de Linde, Sebastian and Sauer, Markus and Kakorin, Sergej and Heilemann, Mike}, journal = {The Journal of Physical Chemistry Letters}, keywords = {rapidstorm}, number = 21, pages = {3156-3159}, title = {Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy}, volume = 1, year = 2010 }

  %0 Journal Article %1 doi:10.1021/jz101300t %A Kottke, Tilman %A van de Linde, Sebastian %A Sauer, Markus %A Kakorin, Sergej %A Heilemann, Mike %D 2010 %J The Journal of Physical Chemistry Letters %N 21 %P 3156-3159 %R 10.1021/jz101300t %T Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy %U http://pubs.acs.org/doi/abs/10.1021/jz101300t %V 1 %X The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.
• Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility. Endesfelder, Ulrike; van de Linde, Sebastian; Wolter, Steve; Sauer, Markus; Heilemann, Mike. In CHEMPHYSCHEM, 11(4), S. 836–840. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.

  @article{Endesfelder2010, abstract = {Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Endesfelder, Ulrike and van de Linde, Sebastian and Wolter, Steve and Sauer, Markus and Heilemann, Mike}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {03}, number = 4, pages = {836-840}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility}, type = {Article}, volume = 11, year = 2010 }

  %0 Journal Article %1 Endesfelder2010 %A Endesfelder, Ulrike %A van de Linde, Sebastian %A Wolter, Steve %A Sauer, Markus %A Heilemann, Mike %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 4 %P 836-840 %T Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility %U http://dx.doi.org/10.1002/cphc.200900944 %V 11 %X Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.
• DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis. Schlereth, Katharina; Beinoraviciute-Kellner, Rasa; Zeitlinger, Marie K.; Bretz, Anne C.; Sauer, Markus; Charles, Joel P.; Vogiatzi, Fotini; Leich, Ellen; Samans, Birgit; Eilers, Martin; Kisker, Caroline; Rosenwald, Andreas; Stiewe, Thorsten. In MOLECULAR CELL, 38(3), S. 356–368. CELL PRESS, 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA, 2010.
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  p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.

  @article{Schlereth2010, abstract = {p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.}, address = {600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA}, author = {Schlereth, Katharina and Beinoraviciute-Kellner, Rasa and Zeitlinger, Marie K. and Bretz, Anne C. and Sauer, Markus and Charles, Joel P. and Vogiatzi, Fotini and Leich, Ellen and Samans, Birgit and Eilers, Martin and Kisker, Caroline and Rosenwald, Andreas and Stiewe, Thorsten}, journal = {MOLECULAR CELL}, keywords = {sauer}, month = {05}, number = 3, pages = {356-368}, publisher = {CELL PRESS}, title = {DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis}, type = {Article}, volume = 38, year = 2010 }

  %0 Journal Article %1 Schlereth2010 %A Schlereth, Katharina %A Beinoraviciute-Kellner, Rasa %A Zeitlinger, Marie K. %A Bretz, Anne C. %A Sauer, Markus %A Charles, Joel P. %A Vogiatzi, Fotini %A Leich, Ellen %A Samans, Birgit %A Eilers, Martin %A Kisker, Caroline %A Rosenwald, Andreas %A Stiewe, Thorsten %C 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA %D 2010 %I CELL PRESS %J MOLECULAR CELL %N 3 %P 356-368 %T DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis %U http://dx.doi.org/10.1016/j.molcel.2010.02.037 %V 38 %X p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.
• Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy. Goehler, Antonia; Andre, Sabine; Kaltner, Herbert; Sauer, Markus; Gabius, Hans-Joachim; Doose, Sören. In BIOPHYSICAL JOURNAL, 98(12), S. 3044–3053. CELL PRESS, 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA, 2010.
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  Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.

  @article{Goehler2010, abstract = {Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.}, address = {600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA}, author = {Goehler, Antonia and Andre, Sabine and Kaltner, Herbert and Sauer, Markus and Gabius, Hans-Joachim and Doose, Sören}, journal = {BIOPHYSICAL JOURNAL}, keywords = {sauer}, month = {06}, number = 12, pages = {3044-3053}, publisher = {CELL PRESS}, title = {Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy}, type = {Article}, volume = 98, year = 2010 }

  %0 Journal Article %1 Goehler2010 %A Goehler, Antonia %A Andre, Sabine %A Kaltner, Herbert %A Sauer, Markus %A Gabius, Hans-Joachim %A Doose, Sören %C 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA %D 2010 %I CELL PRESS %J BIOPHYSICAL JOURNAL %N 12 %P 3044-3053 %T Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy %U http://dx.doi.org/10.1016/j.bpj.2010.03.040 %V 98 %X Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.
• In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET. Seidel, Thorsten; Seefeldt, Britta; Sauer, Markus; Dietz, Karl-Josef. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 272–279. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Seidel2010, abstract = {Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Seidel, Thorsten and Seefeldt, Britta and Sauer, Markus and Dietz, Karl-Josef}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {09}, number = {4, Sp. Iss. SI}, pages = {272-279}, publisher = {ELSEVIER SCIENCE BV}, title = {In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Seidel2010 %A Seidel, Thorsten %A Seefeldt, Britta %A Sauer, Markus %A Dietz, Karl-Josef %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 272-279 %T In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET %U http://dx.doi.org/10.1016/j.jbiotec.2010.06.016 %V 149 %X Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.
• The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. van de Linde, Sebastian; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 260–266. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Linde2010, abstract = {Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {van de Linde, Sebastian and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {mike}, month = {09}, number = {4, Sp. Iss. SI}, pages = {260-266}, publisher = {ELSEVIER SCIENCE BV}, title = {The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Linde2010 %A van de Linde, Sebastian %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 260-266 %T The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging %U http://dx.doi.org/10.1016/j.jbiotec.2010.02.010 %V 149 %X Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.
• Fluorescently labeled 1 nm thin nanomembranes. Nottbohm, Christoph T.; Sopher, Ran; Heilemann, Mike; Sauer, Markus; Goelzhaeuser, Armin. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 267–271. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Nottbohm2010, abstract = {Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Nottbohm, Christoph T. and Sopher, Ran and Heilemann, Mike and Sauer, Markus and Goelzhaeuser, Armin}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {mike}, month = {09}, number = {4, Sp. Iss. SI}, pages = {267-271}, publisher = {ELSEVIER SCIENCE BV}, title = {Fluorescently labeled 1 nm thin nanomembranes}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Nottbohm2010 %A Nottbohm, Christoph T. %A Sopher, Ran %A Heilemann, Mike %A Sauer, Markus %A Goelzhaeuser, Armin %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 267-271 %T Fluorescently labeled 1 nm thin nanomembranes %U http://dx.doi.org/10.1016/j.jbiotec.2010.01.018 %V 149 %X Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.
• BioImaging: Contributions from biology, physics and informatics. Goelzhaeuser, A.; Nattkemper, T.; Niehaus, K.; Puehler, A.; Sauer, M. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 227–228. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  @article{Goelzhaeuser2010, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Goelzhaeuser, A. and Nattkemper, T. and Niehaus, K. and Puehler, A. and Sauer, M.}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {09}, number = {4, Sp. Iss. SI}, pages = {227-228}, publisher = {ELSEVIER SCIENCE BV}, title = {BioImaging: Contributions from biology, physics and informatics}, type = {Editorial Material}, volume = 149, year = 2010 }

  %0 Journal Article %1 Goelzhaeuser2010 %A Goelzhaeuser, A. %A Nattkemper, T. %A Niehaus, K. %A Puehler, A. %A Sauer, M. %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 227-228 %T BioImaging: Contributions from biology, physics and informatics %U http://dx.doi.org/10.1016/j.jbiotec.2010.08.015 %V 149
• Live-cell super-resolution imaging with trimethoprim conjugates. Wombacher, Richard; Heidbreder, Meike; van de Linde, Sebastian; Sheetz, Michael P.; Heilemann, Mike; Cornish, Virginia W.; Sauer, Markus. In NATURE METHODS, 7(9), S. 717-U72. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2010.
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  The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.

  @article{Wombacher2010, abstract = {The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Wombacher, Richard and Heidbreder, Meike and van de Linde, Sebastian and Sheetz, Michael P. and Heilemann, Mike and Cornish, Virginia W. and Sauer, Markus}, journal = {NATURE METHODS}, keywords = {mike}, month = {09}, number = 9, pages = {717-U72}, publisher = {NATURE PUBLISHING GROUP}, title = {Live-cell super-resolution imaging with trimethoprim conjugates}, type = {Article}, volume = 7, year = 2010 }

  %0 Journal Article %1 Wombacher2010 %A Wombacher, Richard %A Heidbreder, Meike %A van de Linde, Sebastian %A Sheetz, Michael P. %A Heilemann, Mike %A Cornish, Virginia W. %A Sauer, Markus %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2010 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 9 %P 717-U72 %T Live-cell super-resolution imaging with trimethoprim conjugates %U http://dx.doi.org/10.1038/NMETH.1489 %V 7 %X The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.

• Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging. van de Linde, Sebastian; Endesfelder, Ulrike; Mukherjee, Anindita; Schuettpelz, Mark; Wiebusch, Gerd; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 8(4), S. 465–469. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2009.
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  We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.

  @article{Linde2009, abstract = {We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {van de Linde, Sebastian and Endesfelder, Ulrike and Mukherjee, Anindita and Schuettpelz, Mark and Wiebusch, Gerd and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {PHOTOCHEMICAL \& PHOTOBIOLOGICAL SCIENCES}, keywords = {mike}, number = 4, pages = {465-469}, publisher = {ROYAL SOC CHEMISTRY}, title = {Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging}, type = {Article}, volume = 8, year = 2009 }

  %0 Journal Article %1 Linde2009 %A van de Linde, Sebastian %A Endesfelder, Ulrike %A Mukherjee, Anindita %A Schuettpelz, Mark %A Wiebusch, Gerd %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2009 %I ROYAL SOC CHEMISTRY %J PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES %N 4 %P 465-469 %T Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging %U http://dx.doi.org/10.1039/b822533h %V 8 %X We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.
• Super-Resolution Imaging with Small Organic Fluorophores. Heilemann, Mike; van de Linde, Sebastian; Mukherjee, Anindita; Sauer, Markits. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 48(37), S. 6903–6908. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.

  @article{Heilemann2009b, abstract = {What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and van de Linde, Sebastian and Mukherjee, Anindita and Sauer, Markits}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 37, pages = {6903-6908}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Super-Resolution Imaging with Small Organic Fluorophores}, type = {Article}, volume = 48, year = 2009 }

  %0 Journal Article %1 Heilemann2009b %A Heilemann, Mike %A van de Linde, Sebastian %A Mukherjee, Anindita %A Sauer, Markits %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 37 %P 6903-6908 %T Super-Resolution Imaging with Small Organic Fluorophores %U http://dx.doi.org/10.1002/anie.200902073 %V 48 %X What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.
• Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification. Heilemann, Mike; Dedecker, Peter; Hofkens, Johan; Sauer, Markus. In LASER & PHOTONICS REVIEWS, 3(1-2), S. 180–202. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.

  @article{Heilemann2009a, abstract = {Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and Dedecker, Peter and Hofkens, Johan and Sauer, Markus}, journal = {LASER \& PHOTONICS REVIEWS}, keywords = {mike}, month = {03}, number = {1-2}, pages = {180-202}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification}, type = {Review}, volume = 3, year = 2009 }

  %0 Journal Article %1 Heilemann2009a %A Heilemann, Mike %A Dedecker, Peter %A Hofkens, Johan %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J LASER & PHOTONICS REVIEWS %N 1-2 %P 180-202 %T Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification %U http://dx.doi.org/10.1002/lpor.200810043 %V 3 %X Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.
• Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution. Hennig, Simon; van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus. In NANO LETTERS, 9(6), S. 2466–2470. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2009.
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  We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.

  @article{Hennig2009, abstract = {We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Hennig, Simon and van de Linde, Sebastian and Heilemann, Mike and Sauer, Markus}, journal = {NANO LETTERS}, keywords = {mike}, month = {06}, number = 6, pages = {2466-2470}, publisher = {AMER CHEMICAL SOC}, title = {Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution}, type = {Article}, volume = 9, year = 2009 }

  %0 Journal Article %1 Hennig2009 %A Hennig, Simon %A van de Linde, Sebastian %A Heilemann, Mike %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2009 %I AMER CHEMICAL SOC %J NANO LETTERS %N 6 %P 2466-2470 %T Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution %U http://dx.doi.org/10.1021/nl9012387 %V 9 %X We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.
• Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules. Doose, Sören; Neuweiler, Hannes; Sauer, Markus. In CHEMPHYSCHEM, 10(9-10, Sp. Iss. SI), S. 1389–1398. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

  @article{Doose2009, abstract = {Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören and Neuweiler, Hannes and Sauer, Markus}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = {07}, number = {9-10, Sp. Iss. SI}, pages = {1389-1398}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules}, type = {Review}, volume = 10, year = 2009 }

  %0 Journal Article %1 Doose2009 %A Doose, Sören %A Neuweiler, Hannes %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 9-10, Sp. Iss. SI %P 1389-1398 %T Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules %U http://dx.doi.org/10.1002/cphc.200900238 %V 10 %X Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

• Photoswitching microscopy with standard fluorophores. van de Linde, S.; Kasper, R.; Heilemann, M.; Sauer, M. In APPLIED PHYSICS B-LASERS AND OPTICS, 93(4), S. 725–731. SPRINGER, 233 SPRING ST, NEW YORK, NY 10013 USA, 2008.
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  We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

  @article{Linde2008, abstract = {We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, author = {van de Linde, S. and Kasper, R. and Heilemann, M. and Sauer, M.}, journal = {APPLIED PHYSICS B-LASERS AND OPTICS}, keywords = {mike}, month = 12, number = 4, pages = {725-731}, publisher = {SPRINGER}, title = {Photoswitching microscopy with standard fluorophores}, type = {Article}, volume = 93, year = 2008 }

  %0 Journal Article %1 Linde2008 %A van de Linde, S. %A Kasper, R. %A Heilemann, M. %A Sauer, M. %C 233 SPRING ST, NEW YORK, NY 10013 USA %D 2008 %I SPRINGER %J APPLIED PHYSICS B-LASERS AND OPTICS %N 4 %P 725-731 %T Photoswitching microscopy with standard fluorophores %U http://dx.doi.org/10.1007/s00340-008-3250-9 %V 93 %X We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.
• Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores. van de Linde, Sebastian; Sauer, Markus; Heilemann, Mike. In JOURNAL OF STRUCTURAL BIOLOGY, 164(3), S. 250–254. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2008.
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  Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.

  @article{Linde2008a, abstract = {Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {van de Linde, Sebastian and Sauer, Markus and Heilemann, Mike}, journal = {JOURNAL OF STRUCTURAL BIOLOGY}, keywords = {mike}, month = 12, number = 3, pages = {250-254}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores}, type = {Article}, volume = 164, year = 2008 }

  %0 Journal Article %1 Linde2008a %A van de Linde, Sebastian %A Sauer, Markus %A Heilemann, Mike %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2008 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J JOURNAL OF STRUCTURAL BIOLOGY %N 3 %P 250-254 %T Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores %U http://dx.doi.org/10.1016/j.jsb.2008.08.002 %V 164 %X Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.
• Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy. Schuettpelz, Mark; Schoening, Jan C.; Doose, Sören; Neuweiler, Hannes; Peters, Elisabeth; Staiger, Dorothee; Sauer, Markus. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 130(29), S. 9507–9513. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2008.
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  The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

  @article{Schuettpelz2008, abstract = {The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Schuettpelz, Mark and Schoening, Jan C. and Doose, Sören and Neuweiler, Hannes and Peters, Elisabeth and Staiger, Dorothee and Sauer, Markus}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {07}, number = 29, pages = {9507-9513}, publisher = {AMER CHEMICAL SOC}, title = {Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 130, year = 2008 }

  %0 Journal Article %1 Schuettpelz2008 %A Schuettpelz, Mark %A Schoening, Jan C. %A Doose, Sören %A Neuweiler, Hannes %A Peters, Elisabeth %A Staiger, Dorothee %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2008 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 29 %P 9507-9513 %T Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ja801994z %V 130 %X The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.
• Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis. Seidel, Thorsten; Schnitzer, Daniel; Golldack, Dortje; Sauer, Markus; Dietz, Karl-Josef. In BMC CELL BIOLOGY, 9. BIOMED CENTRAL LTD, CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND, 2008.
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  Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.

  @article{Seidel2008, abstract = {Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.}, address = {CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND}, author = {Seidel, Thorsten and Schnitzer, Daniel and Golldack, Dortje and Sauer, Markus and Dietz, Karl-Josef}, journal = {BMC CELL BIOLOGY}, keywords = {sauer}, month = {05}, publisher = {BIOMED CENTRAL LTD}, title = {Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis}, type = {Article}, volume = 9, year = 2008 }

  %0 Journal Article %1 Seidel2008 %A Seidel, Thorsten %A Schnitzer, Daniel %A Golldack, Dortje %A Sauer, Markus %A Dietz, Karl-Josef %C CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND %D 2008 %I BIOMED CENTRAL LTD %J BMC CELL BIOLOGY %T Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis %U http://dx.doi.org/10.1186/1471-2121-9-28 %V 9 %X Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.
• Fluorescent proteins for single-molecule fluorescence applications. Seefeldt, Britta; Kasper, Robert; Seidel, Thorsten; Tinnefeld, Philip; Dietz, Karl-Josef; Heilemann, Mike; Sauer, Markus. In JOURNAL OF BIOPHOTONICS, 1(1), S. 74–82. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  @article{Seefeldt2008, abstract = {We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH \& Co. KGaA, Weinheim}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Seefeldt, Britta and Kasper, Robert and Seidel, Thorsten and Tinnefeld, Philip and Dietz, Karl-Josef and Heilemann, Mike and Sauer, Markus}, journal = {JOURNAL OF BIOPHOTONICS}, keywords = {mike}, month = {02}, number = 1, pages = {74-82}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Fluorescent proteins for single-molecule fluorescence applications}, type = {Article}, volume = 1, year = 2008 }

  %0 Journal Article %1 Seefeldt2008 %A Seefeldt, Britta %A Kasper, Robert %A Seidel, Thorsten %A Tinnefeld, Philip %A Dietz, Karl-Josef %A Heilemann, Mike %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J JOURNAL OF BIOPHOTONICS %N 1 %P 74-82 %T Fluorescent proteins for single-molecule fluorescence applications %U http://dx.doi.org/10.1002/jbio.200710024 %V 1 %X We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
• Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Heilemann, Mike; van de Linde, Sebastian; Schuttpelz, Mark; Kasper, Robert; Seefeldt, Britta; Mukherjee, Anindita; Tinnefeld, Philip; Sauer, Markus. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 47(33), S. 6172–6176. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.

  @article{Heilemann2008, abstract = {Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and van de Linde, Sebastian and Schuttpelz, Mark and Kasper, Robert and Seefeldt, Britta and Mukherjee, Anindita and Tinnefeld, Philip and Sauer, Markus}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 33, pages = {6172-6176}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes}, type = {Article}, volume = 47, year = 2008 }

  %0 Journal Article %1 Heilemann2008 %A Heilemann, Mike %A van de Linde, Sebastian %A Schuttpelz, Mark %A Kasper, Robert %A Seefeldt, Britta %A Mukherjee, Anindita %A Tinnefeld, Philip %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 33 %P 6172-6176 %T Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes %U http://dx.doi.org/10.1002/anie.200802376 %V 47 %X Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.
• A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Vogelsang, Jan; Kasper, Robert; Steinhauer, Christian; Person, Britta; Heilemann, Mike; Sauer, Markus; Tinnefeld, Philip. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 47(29), S. 5465–5469. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.

  @article{Vogelsang2008, abstract = {On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Vogelsang, Jan and Kasper, Robert and Steinhauer, Christian and Person, Britta and Heilemann, Mike and Sauer, Markus and Tinnefeld, Philip}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 29, pages = {5465-5469}, publisher = {WILEY-V C H VERLAG GMBH}, title = {A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes}, type = {Article}, volume = 47, year = 2008 }

  %0 Journal Article %1 Vogelsang2008 %A Vogelsang, Jan %A Kasper, Robert %A Steinhauer, Christian %A Person, Britta %A Heilemann, Mike %A Sauer, Markus %A Tinnefeld, Philip %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 29 %P 5465-5469 %T A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes %U http://dx.doi.org/10.1002/anie.200801518 %V 47 %X On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.

• Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. Neuweiler, Hannes; Lollmann, Marc; Doose, Sören; Sauer, Markus. In JOURNAL OF MOLECULAR BIOLOGY, 365(3), S. 856–869. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2007.
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  Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2007, abstract = {Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Lollmann, Marc and Doose, Sören and Sauer, Markus}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {01}, number = 3, pages = {856-869}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 365, year = 2007 }

  %0 Journal Article %1 Neuweiler2007 %A Neuweiler, Hannes %A Lollmann, Marc %A Doose, Sören %A Sauer, Markus %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2007 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 3 %P 856-869 %T Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1016/j.jmb.2006.10.021 %V 365 %X Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.
• Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis. Schulze, Philipp; Schuettpelz, Mark; Sauer, Markus; Belder, Detlev. In LAB ON A CHIP, 7(12), S. 1841–1844. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2007.
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  Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.

  @article{Schulze2007, abstract = {Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {Schulze, Philipp and Schuettpelz, Mark and Sauer, Markus and Belder, Detlev}, journal = {LAB ON A CHIP}, keywords = {sauer}, number = 12, pages = {1841-1844}, publisher = {ROYAL SOC CHEMISTRY}, title = {Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis}, type = {Article}, volume = 7, year = 2007 }

  %0 Journal Article %1 Schulze2007 %A Schulze, Philipp %A Schuettpelz, Mark %A Sauer, Markus %A Belder, Detlev %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2007 %I ROYAL SOC CHEMISTRY %J LAB ON A CHIP %N 12 %P 1841-1844 %T Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis %U http://dx.doi.org/10.1039/b710762e %V 7 %X Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.
• A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer. Agaylan, Ashraf; Binder, Daniel; Sauer, Markus; Neuweiler, Hannes; Meyer, Oliver; Kiesewetter, Holger; Salama, Abdulgabar. In Cancer, 110(11), S. 2502–2506. Wiley Subscription Services, Inc., A Wiley Company, 2007.
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  Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.

  @article{CNCR:CNCR23057, abstract = {Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.}, author = {Agaylan, Ashraf and Binder, Daniel and Sauer, Markus and Neuweiler, Hannes and Meyer, Oliver and Kiesewetter, Holger and Salama, Abdulgabar}, journal = {Cancer}, keywords = {hannes}, number = 11, pages = {2502–2506}, publisher = {Wiley Subscription Services, Inc., A Wiley Company}, title = {A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer}, volume = 110, year = 2007 }

  %0 Journal Article %1 CNCR:CNCR23057 %A Agaylan, Ashraf %A Binder, Daniel %A Sauer, Markus %A Neuweiler, Hannes %A Meyer, Oliver %A Kiesewetter, Holger %A Salama, Abdulgabar %D 2007 %I Wiley Subscription Services, Inc., A Wiley Company %J Cancer %N 11 %P 2502--2506 %R 10.1002/cncr.23057 %T A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer %U http://dx.doi.org/10.1002/cncr.23057 %V 110 %X Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.
• Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix. Doose, Sören; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(44), S. 17400–17405. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2007.
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  Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

  @article{Doose2007c, abstract = {Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Doose, Sören and Neuweiler, Hannes and Barsch, Hannes and Sauer, Markus}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 10, number = 44, pages = {17400-17405}, publisher = {NATL ACAD SCIENCES}, title = {Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix}, type = {Article}, volume = 104, year = 2007 }

  %0 Journal Article %1 Doose2007c %A Doose, Sören %A Neuweiler, Hannes %A Barsch, Hannes %A Sauer, Markus %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2007 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 44 %P 17400-17405 %T Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix %U http://dx.doi.org/10.1073/pnas.0705605104 %V 104 %X Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.
• Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment. Clifford, John N.; Bell, Toby D. M.; Tinnefeld, Philip; Heilemann, Mike; Melnikov, Sergey M.; ichi Hotta, Jun; Sliwa, Michel; Dedecker, Peter; Sauer, Markus; Hofkens, Johan; Yeow, Edwin K. L. In JOURNAL OF PHYSICAL CHEMISTRY B, 111(25), S. 6987–6991. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2007.
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  The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.

  @article{Clifford2007, abstract = {The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Clifford, John N. and Bell, Toby D. M. and Tinnefeld, Philip and Heilemann, Mike and Melnikov, Sergey M. and ichi Hotta, Jun and Sliwa, Michel and Dedecker, Peter and Sauer, Markus and Hofkens, Johan and Yeow, Edwin K. L.}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {mike}, month = {06}, number = 25, pages = {6987-6991}, publisher = {AMER CHEMICAL SOC}, title = {Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment}, type = {Article}, volume = 111, year = 2007 }

  %0 Journal Article %1 Clifford2007 %A Clifford, John N. %A Bell, Toby D. M. %A Tinnefeld, Philip %A Heilemann, Mike %A Melnikov, Sergey M. %A ichi Hotta, Jun %A Sliwa, Michel %A Dedecker, Peter %A Sauer, Markus %A Hofkens, Johan %A Yeow, Edwin K. L. %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2007 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 25 %P 6987-6991 %R 10.1021/jp072864d %T Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment %U http://dx.doi.org/10.1021/jp072864d %V 111 %X The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.
• Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range. Vogelsang, Jan; Doose, Sören; Sauer, Markus; Tinnefeld, Philip. In ANALYTICAL CHEMISTRY, 79(19), S. 7367–7375. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2007.
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  In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.

  @article{Vogelsang2007, abstract = {In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Vogelsang, Jan and Doose, Sören and Sauer, Markus and Tinnefeld, Philip}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 10, number = 19, pages = {7367-7375}, publisher = {AMER CHEMICAL SOC}, title = {Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range}, type = {Article}, volume = 79, year = 2007 }

  %0 Journal Article %1 Vogelsang2007 %A Vogelsang, Jan %A Doose, Sören %A Sauer, Markus %A Tinnefeld, Philip %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2007 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 19 %P 7367-7375 %T Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range %U http://dx.doi.org/10.1021/ac071176n %V 79 %X In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.
• Polymer properties of polythymine as revealed by translational diffusion. Doose, Sören; Barsch, Hannes; Sauer, Markus. In BIOPHYSICAL JOURNAL, 93(4), S. 1224–1234. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2007.
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  Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.

  @article{Doose2007b, abstract = {Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Doose, Sören and Barsch, Hannes and Sauer, Markus}, journal = {BIOPHYSICAL JOURNAL}, keywords = {sauer}, month = {08}, number = 4, pages = {1224-1234}, publisher = {BIOPHYSICAL SOCIETY}, title = {Polymer properties of polythymine as revealed by translational diffusion}, type = {Article}, volume = 93, year = 2007 }

  %0 Journal Article %1 Doose2007b %A Doose, Sören %A Barsch, Hannes %A Sauer, Markus %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2007 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 4 %P 1224-1234 %T Polymer properties of polythymine as revealed by translational diffusion %U http://dx.doi.org/10.1529/biophysj.107.107342 %V 93 %X Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.

• DNA-based molecular wires: Multiple emission pathways of individual constructs. Sanchez-Mosteiro, Gabriel; van Dijk, Erik M. H. P.; Hernando, Jordi; Heilemann, Mike; Tinnefeld, Philip; Sauer, Markus; Koberlin, Felix; Patting, Matthias; Wahl, Michael; Erdmann, Rainer; van Hulst, Niek F.; Garcia-Parajo, Maria F. In JOURNAL OF PHYSICAL CHEMISTRY B, 110(51), S. 26349–26353. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.

  @article{Sanchez-Mosteiro2006, abstract = {The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100\% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Sanchez-Mosteiro, Gabriel and van Dijk, Erik M. H. P. and Hernando, Jordi and Heilemann, Mike and Tinnefeld, Philip and Sauer, Markus and Koberlin, Felix and Patting, Matthias and Wahl, Michael and Erdmann, Rainer and van Hulst, Niek F. and Garcia-Parajo, Maria F.}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {mike}, month = 12, number = 51, pages = {26349-26353}, publisher = {AMER CHEMICAL SOC}, title = {DNA-based molecular wires: Multiple emission pathways of individual constructs}, type = {Article}, volume = 110, year = 2006 }

  %0 Journal Article %1 Sanchez-Mosteiro2006 %A Sanchez-Mosteiro, Gabriel %A van Dijk, Erik M. H. P. %A Hernando, Jordi %A Heilemann, Mike %A Tinnefeld, Philip %A Sauer, Markus %A Koberlin, Felix %A Patting, Matthias %A Wahl, Michael %A Erdmann, Rainer %A van Hulst, Niek F. %A Garcia-Parajo, Maria F. %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 51 %P 26349-26353 %T DNA-based molecular wires: Multiple emission pathways of individual constructs %U http://dx.doi.org/10.1021/jp064701f %V 110 %X The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.
• Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires. Heilemann, Mike; Kasper, Robert; Tinnefeld, Philip; Sauer, Markus. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(51), S. 16864–16875. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.

  @article{Heilemann2006, abstract = {Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, Mike and Kasper, Robert and Tinnefeld, Philip and Sauer, Markus}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {mike}, month = 12, number = 51, pages = {16864-16875}, publisher = {AMER CHEMICAL SOC}, title = {Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires}, type = {Article}, volume = 128, year = 2006 }

  %0 Journal Article %1 Heilemann2006 %A Heilemann, Mike %A Kasper, Robert %A Tinnefeld, Philip %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 51 %P 16864-16875 %T Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires %U http://dx.doi.org/10.1021/ja065585x %V 128 %X Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.
• p53 family members in myogenic differentiation and rhabdomyosarcoma development. Cam, Hakan; Griesmann, Heidi; Beitzinger, Michaela; Hofmann, Lars; Beinoraviciute-Kellner, Rasa; Sauer, Markus; Huettinger-Kirchhof, Nicole; Oswald, Claudia; Friedl, Peter; Gattenloehner, Stefan; Burek, Christof; Rosenwald, Andreas; Stiewe, Thorsten. In CANCER CELL, 10(4), S. 281–293. CELL PRESS, 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA, 2006.
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  The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.

  @article{Cam2006, abstract = {The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.}, address = {1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA}, author = {Cam, Hakan and Griesmann, Heidi and Beitzinger, Michaela and Hofmann, Lars and Beinoraviciute-Kellner, Rasa and Sauer, Markus and Huettinger-Kirchhof, Nicole and Oswald, Claudia and Friedl, Peter and Gattenloehner, Stefan and Burek, Christof and Rosenwald, Andreas and Stiewe, Thorsten}, journal = {CANCER CELL}, keywords = {sauer}, month = 10, number = 4, pages = {281-293}, publisher = {CELL PRESS}, title = {p53 family members in myogenic differentiation and rhabdomyosarcoma development}, type = {Article}, volume = 10, year = 2006 }

  %0 Journal Article %1 Cam2006 %A Cam, Hakan %A Griesmann, Heidi %A Beitzinger, Michaela %A Hofmann, Lars %A Beinoraviciute-Kellner, Rasa %A Sauer, Markus %A Huettinger-Kirchhof, Nicole %A Oswald, Claudia %A Friedl, Peter %A Gattenloehner, Stefan %A Burek, Christof %A Rosenwald, Andreas %A Stiewe, Thorsten %C 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA %D 2006 %I CELL PRESS %J CANCER CELL %N 4 %P 281-293 %T p53 family members in myogenic differentiation and rhabdomyosarcoma development %U http://dx.doi.org/10.1016/j.ccr.2006.08.024 %V 10 %X The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.
• Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals. Biebricher, A; Sauer, M; Tinnefeld, P. In JOURNAL OF PHYSICAL CHEMISTRY B, 110(11), S. 5174–5178. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.

  @article{Biebricher2006, abstract = {Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Biebricher, A and Sauer, M and Tinnefeld, P}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = {03}, number = 11, pages = {5174-5178}, publisher = {AMER CHEMICAL SOC}, title = {Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals}, type = {Letter}, volume = 110, year = 2006 }

  %0 Journal Article %1 Biebricher2006 %A Biebricher, A %A Sauer, M %A Tinnefeld, P %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 11 %P 5174-5178 %T Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals %U http://dx.doi.org/10.1021/jp060660b %V 110 %X Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.
• Application of multiline two-photon microscopy to functional in vivo imaging. Kurtz, R; Fricke, M; Kalb, J; Tinnefeld, P; Sauer, M. In JOURNAL OF NEUROSCIENCE METHODS, 151(2), S. 276–286. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2006.
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  High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.

  @article{Kurtz2006, abstract = {High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Kurtz, R and Fricke, M and Kalb, J and Tinnefeld, P and Sauer, M}, journal = {JOURNAL OF NEUROSCIENCE METHODS}, keywords = {sauer}, month = {03}, number = 2, pages = {276-286}, publisher = {ELSEVIER SCIENCE BV}, title = {Application of multiline two-photon microscopy to functional in vivo imaging}, type = {Article}, volume = 151, year = 2006 }

  %0 Journal Article %1 Kurtz2006 %A Kurtz, R %A Fricke, M %A Kalb, J %A Tinnefeld, P %A Sauer, M %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2006 %I ELSEVIER SCIENCE BV %J JOURNAL OF NEUROSCIENCE METHODS %N 2 %P 276-286 %T Application of multiline two-photon microscopy to functional in vivo imaging %U http://dx.doi.org/10.1016/j.jneumeth.2005.12.003 %V 151 %X High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.
• Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Marmé, Nicole; Friedrich, Achim; Müller, Matthias; Nolte, Oliver; Wolfrum, Jürgen; Hoheisel, Jörg D.; Sauer, Markus; Knemeyer, Jens-Peter. In Nucl. Acids Res., 34(13), S. e90. 2006.
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  We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s

  @article{marméidentification, abstract = {We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s}, author = {Marmé, Nicole and Friedrich, Achim and Müller, Matthias and Nolte, Oliver and Wolfrum, Jürgen and Hoheisel, Jörg D. and Sauer, Markus and Knemeyer, Jens-Peter}, journal = {Nucl. Acids Res.}, keywords = {sauer}, number = 13, pages = {e90}, title = {Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy}, volume = 34, year = 2006 }

  %0 Journal Article %1 marméidentification %A Marmé, Nicole %A Friedrich, Achim %A Müller, Matthias %A Nolte, Oliver %A Wolfrum, Jürgen %A Hoheisel, Jörg D. %A Sauer, Markus %A Knemeyer, Jens-Peter %D 2006 %J Nucl. Acids Res. %N 13 %P e90 %T Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy %U http://nar.oxfordjournals.org/content/34/13/e90 %V 34 %X We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s
• UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions. Schüttpelz, Mark; Müller, Christian; Neuweiler, Hannes; Sauer, Markus. In Analytical Chemistry, 78(3), S. 663–669. 2006.
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  Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays

  @article{doi:10.1021/ac051938j, abstract = {Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays}, author = {Schüttpelz, Mark and Müller, Christian and Neuweiler, Hannes and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {hannes}, note = {PMID: 16448037}, number = 3, pages = {663-669}, title = {UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions}, volume = 78, year = 2006 }

  %0 Journal Article %1 doi:10.1021/ac051938j %A Schüttpelz, Mark %A Müller, Christian %A Neuweiler, Hannes %A Sauer, Markus %D 2006 %J Analytical Chemistry %N 3 %P 663-669 %R 10.1021/ac051938j %T UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions %U http://pubs.acs.org/doi/abs/10.1021/ac051938j %V 78 %X Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays
• The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy. Kim, J; Doose, S; Neuweiler, H; Sauer, M. In NUCLEIC ACIDS RESEARCH, 34(9), S. 2516–2527. OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2006.
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  Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

  @article{Kim2006, abstract = {Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.}, address = {GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND}, author = {Kim, J and Doose, S and Neuweiler, H and Sauer, M}, journal = {NUCLEIC ACIDS RESEARCH}, keywords = {hannes}, number = 9, pages = {2516-2527}, publisher = {OXFORD UNIV PRESS}, title = {The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy}, type = {Article}, volume = 34, year = 2006 }

  %0 Journal Article %1 Kim2006 %A Kim, J %A Doose, S %A Neuweiler, H %A Sauer, M %C GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND %D 2006 %I OXFORD UNIV PRESS %J NUCLEIC ACIDS RESEARCH %N 9 %P 2516-2527 %T The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy %U http://dx.doi.org/10.1093/nar/gkl221 %V 34 %X Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

• High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. Heinlein, T; Biebricher, A; Schluter, P; Roth, CM; Herten, DP; Wolfrum, J; Heilemann, M; Muller, C; Tinnefeld, P; Sauer, M. In CHEMPHYSCHEM, 6(5), S. 949–955. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
  • [ Abstract ]
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  To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

  @article{Heinlein2005, abstract = {To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heinlein, T and Biebricher, A and Schluter, P and Roth, CM and Herten, DP and Wolfrum, J and Heilemann, M and Muller, C and Tinnefeld, P and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {05}, number = 5, pages = {949-955}, publisher = {WILEY-V C H VERLAG GMBH}, title = {High-resolution colocalization of single molecules within the resolution gap of far-field microscopy}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Heinlein2005 %A Heinlein, T %A Biebricher, A %A Schluter, P %A Roth, CM %A Herten, DP %A Wolfrum, J %A Heilemann, M %A Muller, C %A Tinnefeld, P %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 5 %P 949-955 %T High-resolution colocalization of single molecules within the resolution gap of far-field microscopy %U http://dx.doi.org/10.1002/cphc.200400622 %V 6 %X To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.
• Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology. Tinnefeld, Philip; Sauer, Markus. In Angewandte Chemie International Edition, 44(18), S. 2642–2671. WILEY-VCH Verlag, 2005.
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  In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.

  @article{ANIE:ANIE200300647, abstract = {In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.}, author = {Tinnefeld, Philip and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, number = 18, pages = {2642–2671}, publisher = {WILEY-VCH Verlag}, title = {Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology}, volume = 44, year = 2005 }

  %0 Journal Article %1 ANIE:ANIE200300647 %A Tinnefeld, Philip %A Sauer, Markus %D 2005 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 18 %P 2642--2671 %R 10.1002/anie.200300647 %T Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology %U http://dx.doi.org/10.1002/anie.200300647 %V 44 %X In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.
• Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy. Scheffler, S; Sauer, M; Neuweiler, H. In ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS, 219(5), S. 665–678. R OLDENBOURG VERLAG, LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY, 2005.
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  We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.

  @article{Scheffler2005, abstract = {We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.}, address = {LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY}, author = {Scheffler, S and Sauer, M and Neuweiler, H}, journal = {ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY \& CHEMICAL PHYSICS}, keywords = {hannes}, number = 5, pages = {665-678}, publisher = {R OLDENBOURG VERLAG}, title = {Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 219, year = 2005 }

  %0 Journal Article %1 Scheffler2005 %A Scheffler, S %A Sauer, M %A Neuweiler, H %C LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY %D 2005 %I R OLDENBOURG VERLAG %J ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS %N 5 %P 665-678 %T Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy %U http://dx.doi.org/10.1524/zpch.219.5.665.64323 %V 219 %X We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.
• Design of molecular photonic wires based on multistep electronic excitation transfer. Tinnefeld, P; Heilemann, M; Sauer, M. In CHEMPHYSCHEM, 6(2), S. 217–222. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
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  Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.

  @article{Tinnefeld2005, abstract = {Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Tinnefeld, P and Heilemann, M and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {02}, number = 2, pages = {217-222}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Design of molecular photonic wires based on multistep electronic excitation transfer}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Tinnefeld2005 %A Tinnefeld, P %A Heilemann, M %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 2 %P 217-222 %T Design of molecular photonic wires based on multistep electronic excitation transfer %U http://dx.doi.org/10.1002/cphc.200400513 %V 6 %X Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.
• Carbocyanine dyes as efficient reversible single-molecule optical switch. Heilemann, M; Margeat, E; Kasper, R; Sauer, M; Tinnefeld, P. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 127(11), S. 3801–3806. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of > 90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.

  @article{Heilemann2005, abstract = {We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher \β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of \> 90\% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, M and Margeat, E and Kasper, R and Sauer, M and Tinnefeld, P}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {mike}, month = {03}, number = 11, pages = {3801-3806}, publisher = {AMER CHEMICAL SOC}, title = {Carbocyanine dyes as efficient reversible single-molecule optical switch}, type = {Article}, volume = 127, year = 2005 }

  %0 Journal Article %1 Heilemann2005 %A Heilemann, M %A Margeat, E %A Kasper, R %A Sauer, M %A Tinnefeld, P %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 11 %P 3801-3806 %T Carbocyanine dyes as efficient reversible single-molecule optical switch %U http://dx.doi.org/10.1021/ja044686x %V 127 %X We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of > 90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.
• Self-quenching DNA probes based on dye dimerization for identification of mycobacteria. Knemeyer, JP; Marme, N; Hafner, B; Habl, G; Schafer, G; Muller, M; Nolte, O; Sauer, M; Wolfrum, J. In INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY, 85(9-11), S. 625–637. TAYLOR & FRANCIS LTD, 4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND, 2005.
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  This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.

  @article{Knemeyer2005, abstract = {This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.}, address = {4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND}, author = {Knemeyer, JP and Marme, N and Hafner, B and Habl, G and Schafer, G and Muller, M and Nolte, O and Sauer, M and Wolfrum, J}, journal = {INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = {08}, number = {9-11}, pages = {625-637}, publisher = {TAYLOR \& FRANCIS LTD}, title = {Self-quenching DNA probes based on dye dimerization for identification of mycobacteria}, type = {Proceedings Paper}, volume = 85, year = 2005 }

  %0 Journal Article %1 Knemeyer2005 %A Knemeyer, JP %A Marme, N %A Hafner, B %A Habl, G %A Schafer, G %A Muller, M %A Nolte, O %A Sauer, M %A Wolfrum, J %C 4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND %D 2005 %I TAYLOR & FRANCIS LTD %J INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY %N 9-11 %P 625-637 %T Self-quenching DNA probes based on dye dimerization for identification of mycobacteria %U http://dx.doi.org/10.1080/03067310500146094 %V 85 %X This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.
• Exploring life by single-molecule fluorescence spectroscopy. Neuweiler, H; Sauer, M. In ANALYTICAL CHEMISTRY, 77(9), S. 178A-185A. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  @article{Neuweiler2005a, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {hannes}, month = {05}, number = 9, pages = {178A-185A}, publisher = {AMER CHEMICAL SOC}, title = {Exploring life by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Neuweiler2005a %A Neuweiler, H %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 9 %P 178A-185A %T Exploring life by single-molecule fluorescence spectroscopy %V 77
• Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging. Sauer, M. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(27), S. 9433–9434. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2005.
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  @article{Sauer2005, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Sauer, M}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {sauer}, month = {07}, number = 27, pages = {9433-9434}, publisher = {NATL ACAD SCIENCES}, title = {Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging}, type = {Editorial Material}, volume = 102, year = 2005 }

  %0 Journal Article %1 Sauer2005 %A Sauer, M %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2005 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 27 %P 9433-9434 %T Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging %U http://dx.doi.org/10.1073/pnas.0504264102 %V 102
• Defocused imaging of quantum-dot angular distribution of radiation. Patra, D; Gregor, I; Enderlein, J; Sauer, M. In APPLIED PHYSICS LETTERS, 87(10). AMER INST PHYSICS, CIRCULATION & FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA, 2005.
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  We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.

  @article{Patra2005, abstract = {We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.}, address = {CIRCULATION \& FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA}, author = {Patra, D and Gregor, I and Enderlein, J and Sauer, M}, journal = {APPLIED PHYSICS LETTERS}, keywords = {sauer}, month = {09}, number = 10, publisher = {AMER INST PHYSICS}, title = {Defocused imaging of quantum-dot angular distribution of radiation}, type = {Article}, volume = 87, year = 2005 }

  %0 Journal Article %1 Patra2005 %A Patra, D %A Gregor, I %A Enderlein, J %A Sauer, M %C CIRCULATION & FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA %D 2005 %I AMER INST PHYSICS %J APPLIED PHYSICS LETTERS %N 10 %T Defocused imaging of quantum-dot angular distribution of radiation %U http://dx.doi.org/10.1063/1.2037194 %V 87 %X We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.
• A close look at fluorescence quenching of organic dyes by tryptophan. Doose, S; Neuweiler, H; Sauer, M. In CHEMPHYSCHEM, 6(11), S. 2277–2285. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
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  Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.

  @article{Doose2005a, abstract = {Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30\% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, S and Neuweiler, H and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = 11, number = 11, pages = {2277-2285}, publisher = {WILEY-V C H VERLAG GMBH}, title = {A close look at fluorescence quenching of organic dyes by tryptophan}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Doose2005a %A Doose, S %A Neuweiler, H %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 11 %P 2277-2285 %T A close look at fluorescence quenching of organic dyes by tryptophan %U http://dx.doi.org/10.1002/cphc.200500191 %V 6 %X Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.
• Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes. Stohr, K; Hafner, B; Nolte, O; Wolfrum, J; Sauer, M; Herten, DP. In ANALYTICAL CHEMISTRY, 77(22), S. 7195–7203. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.

  @article{Stohr2005, abstract = {Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Stohr, K and Hafner, B and Nolte, O and Wolfrum, J and Sauer, M and Herten, DP}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 11, number = 22, pages = {7195-7203}, publisher = {AMER CHEMICAL SOC}, title = {Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Stohr2005 %A Stohr, K %A Hafner, B %A Nolte, O %A Wolfrum, J %A Sauer, M %A Herten, DP %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 22 %P 7195-7203 %T Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes %U http://dx.doi.org/10.1021/ac051447z %V 77 %X Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.
• A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. Neuweiler, H; Doose, S; Sauer, M. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(46), S. 16650–16655. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2005.
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  The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.

  @article{Neuweiler2005, abstract = {The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, H and Doose, S and Sauer, M}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 11, number = 46, pages = {16650-16655}, publisher = {NATL ACAD SCIENCES}, title = {A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate}, type = {Article}, volume = 102, year = 2005 }

  %0 Journal Article %1 Neuweiler2005 %A Neuweiler, H %A Doose, S %A Sauer, M %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2005 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 46 %P 16650-16655 %T A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate %U http://dx.doi.org/10.1073/pnas.0507351102 %V 102 %X The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.

• Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer. Marmé, Nicole; Knemeyer, Jens-Peter; Wolfrum, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, 43(29), S. 3798–3801. WILEY-VCH Verlag, 2004.
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  Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range

  @article{ANIE:ANIE200453835, abstract = {Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range}, author = {Marmé, Nicole and Knemeyer, Jens-Peter and Wolfrum, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, number = 29, pages = {3798–3801}, publisher = {WILEY-VCH Verlag}, title = {Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer}, volume = 43, year = 2004 }

  %0 Journal Article %1 ANIE:ANIE200453835 %A Marmé, Nicole %A Knemeyer, Jens-Peter %A Wolfrum, Jürgen %A Sauer, Markus %D 2004 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 29 %P 3798--3801 %R 10.1002/anie.200453835 %T Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer %U http://dx.doi.org/10.1002/anie.200453835 %V 43 %X Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range
• Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes. Park, Chong-Woo; Knemeyer, Jens-Peter; Marmé, Nicole; Möller, Martin; Spatz, Joachim; Wolfrum, Jürgen; Sauer, Markus. In Chemical Physics, 301(1), S. 105–110. 2004.
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  There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.

  @article{Park2004105, abstract = {There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.}, author = {Park, Chong-Woo and Knemeyer, Jens-Peter and Marmé, Nicole and Möller, Martin and Spatz, Joachim and Wolfrum, Jürgen and Sauer, Markus}, journal = {Chemical Physics}, keywords = {sauer}, number = 1, pages = {105 - 110}, title = {Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes}, volume = 301, year = 2004 }

  %0 Journal Article %1 Park2004105 %A Park, Chong-Woo %A Knemeyer, Jens-Peter %A Marmé, Nicole %A Möller, Martin %A Spatz, Joachim %A Wolfrum, Jürgen %A Sauer, Markus %D 2004 %J Chemical Physics %N 1 %P 105 - 110 %R DOI: 10.1016/j.chemphys.2004.03.006 %T Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes %U http://www.sciencedirect.com/science/article/B6TFM-4C47PSJ-3/2/01a24087fcf29f6dd86fa8d9234396e6 %V 301 %X There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.
• How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions. Tinnefeld, P.; Buschmann, V.; Weston, K. D.; Biebricher, A.; Herten, D. P.; Piestert, O.; Heinlein, T.; Heilemann, M.; Sauer, M. In Recent Res. Devel. Phys. Chem. 7, 95-125. 2004.
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  A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques

  @article{tinnefeld2004single, abstract = {A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques}, author = {Tinnefeld, P. and Buschmann, V. and Weston, K. D. and Biebricher, A. and Herten, D. P. and Piestert, O. and Heinlein, T. and Heilemann, M. and Sauer, M.}, journal = {Recent Res. Devel. Phys. Chem. 7, 95-125.}, keywords = {mike}, title = {How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions}, year = 2004 }

  %0 Journal Article %1 tinnefeld2004single %A Tinnefeld, P. %A Buschmann, V. %A Weston, K. D. %A Biebricher, A. %A Herten, D. P. %A Piestert, O. %A Heinlein, T. %A Heilemann, M. %A Sauer, M. %D 2004 %J Recent Res. Devel. Phys. Chem. 7, 95-125. %T How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions %X A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques
• Single photon emission from a dendrimer containing eight perylene diimide chromophores. Bell, TDM; Habuchi, S; Masuo, S; Osterling, I; Mullen, K; Tinnefeld, P; Sauer, M; van der Auweraer, M; Hofkens, J; Schryver, FC De. In AUSTRALIAN JOURNAL OF CHEMISTRY, 57(12), S. 1169–1173. C S I R O PUBLISHING, 150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA, 2004.
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  A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.

  @article{Bell2004, abstract = {A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.}, address = {150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA}, author = {Bell, TDM and Habuchi, S and Masuo, S and Osterling, I and Mullen, K and Tinnefeld, P and Sauer, M and van der Auweraer, M and Hofkens, J and Schryver, FC De}, journal = {AUSTRALIAN JOURNAL OF CHEMISTRY}, keywords = {sauer}, number = 12, pages = {1169-1173}, publisher = {C S I R O PUBLISHING}, title = {Single photon emission from a dendrimer containing eight perylene diimide chromophores}, type = {Proceedings Paper}, volume = 57, year = 2004 }

  %0 Journal Article %1 Bell2004 %A Bell, TDM %A Habuchi, S %A Masuo, S %A Osterling, I %A Mullen, K %A Tinnefeld, P %A Sauer, M %A van der Auweraer, M %A Hofkens, J %A Schryver, FC De %C 150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA %D 2004 %I C S I R O PUBLISHING %J AUSTRALIAN JOURNAL OF CHEMISTRY %N 12 %P 1169-1173 %T Single photon emission from a dendrimer containing eight perylene diimide chromophores %U http://dx.doi.org/10.1071/CH04133 %V 57 %X A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.
• Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy. Tinnefeld, Philip; Hofkens, Johan; Herten, Dirk-Peter; Masuo, Sadahiro; Vosch, Tom; Cotlet, Mircea; Habuchi, Satoshi; Müllen, Klaus; Schryver, Frans C. De; Sauer, Markus. In ChemPhysChem, 5(11), S. 1786–1790. WILEY-VCH Verlag, 2004.
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  Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems

  @article{CPHC:CPHC200400325, abstract = {Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems}, author = {Tinnefeld, Philip and Hofkens, Johan and Herten, Dirk-Peter and Masuo, Sadahiro and Vosch, Tom and Cotlet, Mircea and Habuchi, Satoshi and Müllen, Klaus and Schryver, Frans C. De and Sauer, Markus}, journal = {ChemPhysChem}, keywords = {sauer}, number = 11, pages = {1786–1790}, publisher = {WILEY-VCH Verlag}, title = {Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy}, volume = 5, year = 2004 }

  %0 Journal Article %1 CPHC:CPHC200400325 %A Tinnefeld, Philip %A Hofkens, Johan %A Herten, Dirk-Peter %A Masuo, Sadahiro %A Vosch, Tom %A Cotlet, Mircea %A Habuchi, Satoshi %A Müllen, Klaus %A Schryver, Frans C. De %A Sauer, Markus %D 2004 %I WILEY-VCH Verlag %J ChemPhysChem %N 11 %P 1786--1790 %R 10.1002/cphc.200400325 %T Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy %U http://dx.doi.org/10.1002/cphc.200400325 %V 5 %X Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems
• Chemical and Biological Investigations of beta-Oligoarginines. Seebach, Dieter; Namoto, Kenji; Mahajan, Yogesh R.; Bindschädler, Pascal; Sustmann, Reiner; Kirsch, Michael; Ryder, Neil S.; Weiss, Markus; Sauer, Markus; Roth, Christian; Werner, Sabine; Beer, Hans-Dietmar; Munding, Christine; Walde, Peter; Voser, Matthias. In Chemistry & Biodiversity, 1(1), S. 65–97. WILEY-VCH Verlag, 2004.
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  In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.

  @article{CBDV:CBDV200490014, abstract = {In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.}, author = {Seebach, Dieter and Namoto, Kenji and Mahajan, Yogesh R. and Bindschädler, Pascal and Sustmann, Reiner and Kirsch, Michael and Ryder, Neil S. and Weiss, Markus and Sauer, Markus and Roth, Christian and Werner, Sabine and Beer, Hans-Dietmar and Munding, Christine and Walde, Peter and Voser, Matthias}, journal = {Chemistry & Biodiversity}, keywords = {sauer}, number = 1, pages = {65–97}, publisher = {WILEY-VCH Verlag}, title = {Chemical and Biological Investigations of beta-Oligoarginines}, volume = 1, year = 2004 }

  %0 Journal Article %1 CBDV:CBDV200490014 %A Seebach, Dieter %A Namoto, Kenji %A Mahajan, Yogesh R. %A Bindschädler, Pascal %A Sustmann, Reiner %A Kirsch, Michael %A Ryder, Neil S. %A Weiss, Markus %A Sauer, Markus %A Roth, Christian %A Werner, Sabine %A Beer, Hans-Dietmar %A Munding, Christine %A Walde, Peter %A Voser, Matthias %D 2004 %I WILEY-VCH Verlag %J Chemistry & Biodiversity %N 1 %P 65--97 %R 10.1002/cbdv.200490014 %T Chemical and Biological Investigations of beta-Oligoarginines %U http://dx.doi.org/10.1002/cbdv.200490014 %V 1 %X In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.
• Optical single molecule techniques for analytical and biological applications. Dirk-Peter, HERTEN; Andreas, BIEBRICHER; Mike, HEILEMANN; Thomas, HEINLEIN; Christian, MÜLLER; Pia, SCHLÜTER; Philip, TINNEFELD; D., WESTON Kenneth; Markus, SAUER; Jürgen, WOLFRUM. In Recent Res. Devel. Applied Phys., Vol. 7 (2004), Part II, S. 345–368. 2004.
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  @article{Dirk-Peter2004, author = {Dirk-Peter, HERTEN and Andreas, BIEBRICHER and Mike, HEILEMANN and Thomas, HEINLEIN and Christian, MÜLLER and Pia, SCHLÜTER and Philip, TINNEFELD and D., WESTON Kenneth and Markus, SAUER and Jürgen, WOLFRUM}, journal = {Recent Res. Devel. Applied Phys.}, keywords = {mike}, pages = {345-368}, title = {Optical single molecule techniques for analytical and biological applications}, volume = {Vol. 7 (2004), Part II}, year = 2004 }

  %0 Journal Article %1 Dirk-Peter2004 %A Dirk-Peter, HERTEN %A Andreas, BIEBRICHER %A Mike, HEILEMANN %A Thomas, HEINLEIN %A Christian, MÜLLER %A Pia, SCHLÜTER %A Philip, TINNEFELD %A D., WESTON Kenneth %A Markus, SAUER %A Jürgen, WOLFRUM %D 2004 %J Recent Res. Devel. Applied Phys. %P 345-368 %T Optical single molecule techniques for analytical and biological applications %V Vol. 7 (2004), Part II
• Multistep Energy Transfer in Single Molecular Photonic Wires. Heilemann, Mike; Tinnefeld, Philip; Mosteiro, Gabriel Sanchez; Parajo, Maria Garcia; Hulst, Niek F. Van; Sauer, Markus. In Journal of the American Chemical Society, 126(21), S. 6514–6515. 2004.
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  We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events

  @article{doi:10.1021/ja049351u, abstract = {We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events}, author = {Heilemann, Mike and Tinnefeld, Philip and Mosteiro, Gabriel Sanchez and Parajo, Maria Garcia and Hulst, Niek F. Van and Sauer, Markus}, journal = {Journal of the American Chemical Society}, keywords = {mike}, number = 21, pages = {6514-6515}, title = {Multistep Energy Transfer in Single Molecular Photonic Wires}, volume = 126, year = 2004 }

  %0 Journal Article %1 doi:10.1021/ja049351u %A Heilemann, Mike %A Tinnefeld, Philip %A Mosteiro, Gabriel Sanchez %A Parajo, Maria Garcia %A Hulst, Niek F. Van %A Sauer, Markus %D 2004 %J Journal of the American Chemical Society %N 21 %P 6514-6515 %R 10.1021/ja049351u %T Multistep Energy Transfer in Single Molecular Photonic Wires %U http://pubs.acs.org/doi/abs/10.1021/ja049351u %V 126 %X We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events
• Implementation of neural networks for the identification of single molecules. Bowen, BP; Scruggs, A; Enderlein, J; Sauer, M; Woodbury, N. In JOURNAL OF PHYSICAL CHEMISTRY A, 108(21), S. 4799–4804. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2004.
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  The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.

  @article{Bowen2004, abstract = {The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Bowen, BP and Scruggs, A and Enderlein, J and Sauer, M and Woodbury, N}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {05}, number = 21, pages = {4799-4804}, publisher = {AMER CHEMICAL SOC}, title = {Implementation of neural networks for the identification of single molecules}, type = {Article}, volume = 108, year = 2004 }

  %0 Journal Article %1 Bowen2004 %A Bowen, BP %A Scruggs, A %A Enderlein, J %A Sauer, M %A Woodbury, N %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2004 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 21 %P 4799-4804 %T Implementation of neural networks for the identification of single molecules %U http://dx.doi.org/10.1021/jp036456v %V 108 %X The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.
• The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP. Brecht, M; Sewald, K; Schiene, K; Keen, G; Fricke, M; Sauer, M; Niehaus, K. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 151–164. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Brecht2004, abstract = {Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100\% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Brecht, M and Sewald, K and Schiene, K and Keen, G and Fricke, M and Sauer, M and Niehaus, K}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {151-164}, publisher = {ELSEVIER SCIENCE BV}, title = {The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Brecht2004 %A Brecht, M %A Sewald, K %A Schiene, K %A Keen, G %A Fricke, M %A Sauer, M %A Niehaus, K %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 151-164 %T The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP %U http://dx.doi.org/10.1016/j.jbiotec.2004.04.030 %V 112 %X Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.
• Multichromophoric dendrimers as single-photon sources: A single-molecule study. Masuo, S; Vosch, T; Cotlet, M; Tinnefeld, P; Habuchi, S; Bell, TDM; Oesterling, I; Beljonne, D; Champagne, B; Mullen, K; Sauer, M; Hofkens, J; Schryver, FC De. In JOURNAL OF PHYSICAL CHEMISTRY B, 108(43), S. 16686–16696. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2004.
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  Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.

  @article{Masuo2004, abstract = {Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Masuo, S and Vosch, T and Cotlet, M and Tinnefeld, P and Habuchi, S and Bell, TDM and Oesterling, I and Beljonne, D and Champagne, B and Mullen, K and Sauer, M and Hofkens, J and Schryver, FC De}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = 10, number = 43, pages = {16686-16696}, publisher = {AMER CHEMICAL SOC}, title = {Multichromophoric dendrimers as single-photon sources: A single-molecule study}, type = {Article}, volume = 108, year = 2004 }

  %0 Journal Article %1 Masuo2004 %A Masuo, S %A Vosch, T %A Cotlet, M %A Tinnefeld, P %A Habuchi, S %A Bell, TDM %A Oesterling, I %A Beljonne, D %A Champagne, B %A Mullen, K %A Sauer, M %A Hofkens, J %A Schryver, FC De %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2004 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 43 %P 16686-16696 %T Multichromophoric dendrimers as single-photon sources: A single-molecule study %U http://dx.doi.org/10.1021/jp047804b %V 108 %X Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.
• Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level. Neuweiler, H; Sauer, M. In CURRENT PHARMACEUTICAL BIOTECHNOLOGY, 5(3), S. 285–298. BENTHAM SCIENCE PUBL LTD, EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES, 2004.
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  This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

  @article{Neuweiler2004, abstract = {This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.}, address = {EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES}, author = {Neuweiler, H and Sauer, M}, journal = {CURRENT PHARMACEUTICAL BIOTECHNOLOGY}, keywords = {hannes}, month = {06}, number = 3, pages = {285-298}, publisher = {BENTHAM SCIENCE PUBL LTD}, title = {Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level}, type = {Review}, volume = 5, year = 2004 }

  %0 Journal Article %1 Neuweiler2004 %A Neuweiler, H %A Sauer, M %C EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES %D 2004 %I BENTHAM SCIENCE PUBL LTD %J CURRENT PHARMACEUTICAL BIOTECHNOLOGY %N 3 %P 285-298 %T Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level %U http://dx.doi.org/10.2174/1389201043376896 %V 5 %X This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.
• Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells. Seidel, T; Kluge, C; Hanitzsch, M; Ross, J; Sauer, M; Dietz, KJ; Golldack, D. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 165–175. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Seidel2004, abstract = {The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Seidel, T and Kluge, C and Hanitzsch, M and Ross, J and Sauer, M and Dietz, KJ and Golldack, D}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {165-175}, publisher = {ELSEVIER SCIENCE BV}, title = {Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Seidel2004 %A Seidel, T %A Kluge, C %A Hanitzsch, M %A Ross, J %A Sauer, M %A Dietz, KJ %A Golldack, D %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 165-175 %T Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells %U http://dx.doi.org/10.1016/j.jbiotec.2004.04.027 %V 112 %X The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.
• Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces. Biebricher, A; Paul, A; Tinnefeld, P; Golzhauser, A; Sauer, M. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 97–107. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Biebricher2004, abstract = {We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Biebricher, A and Paul, A and Tinnefeld, P and Golzhauser, A and Sauer, M}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {97-107}, publisher = {ELSEVIER SCIENCE BV}, title = {Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Biebricher2004 %A Biebricher, A %A Paul, A %A Tinnefeld, P %A Golzhauser, A %A Sauer, M %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 97-107 %T Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces %U http://dx.doi.org/10.1016/j.jbiotec.2004.03.019 %V 112 %X We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.
• Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex. Kluge, C; Seidel, T; Bolte, S; Sharma, SS; Hanitzsch, M; Satiat-Jeunemaitre, B; Ross, J; Sauer, M; Golldack, D; Dietz, KJ. In BMC CELL BIOLOGY, 5. BIOMED CENTRAL LTD, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND, 2004.
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  Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

  @article{Kluge2004, abstract = {Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.}, address = {MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND}, author = {Kluge, C and Seidel, T and Bolte, S and Sharma, SS and Hanitzsch, M and Satiat-Jeunemaitre, B and Ross, J and Sauer, M and Golldack, D and Dietz, KJ}, journal = {BMC CELL BIOLOGY}, keywords = {sauer}, month = {08}, publisher = {BIOMED CENTRAL LTD}, title = {Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex}, type = {Article}, volume = 5, year = 2004 }

  %0 Journal Article %1 Kluge2004 %A Kluge, C %A Seidel, T %A Bolte, S %A Sharma, SS %A Hanitzsch, M %A Satiat-Jeunemaitre, B %A Ross, J %A Sauer, M %A Golldack, D %A Dietz, KJ %C MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND %D 2004 %I BIOMED CENTRAL LTD %J BMC CELL BIOLOGY %T Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex %U http://dx.doi.org/10.1186/1471-2121-5-29 %V 5 %X Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

• Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA. Adhikary, A; Buschmann, V; Muller, C; Sauer, M. In NUCLEIC ACIDS RESEARCH, 31(8), S. 2178–2186. OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2003.
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  Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.

  @article{Adhikary2003, abstract = {Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.}, address = {GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND}, author = {Adhikary, A and Buschmann, V and Muller, C and Sauer, M}, journal = {NUCLEIC ACIDS RESEARCH}, keywords = {sauer}, month = {04}, number = 8, pages = {2178-2186}, publisher = {OXFORD UNIV PRESS}, title = {Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA}, type = {Article}, volume = 31, year = 2003 }

  %0 Journal Article %1 Adhikary2003 %A Adhikary, A %A Buschmann, V %A Muller, C %A Sauer, M %C GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND %D 2003 %I OXFORD UNIV PRESS %J NUCLEIC ACIDS RESEARCH %N 8 %P 2178-2186 %T Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA %U http://dx.doi.org/10.1093/nar/gkg308 %V 31 %X Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.
• Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer. Sauer, M. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 42(16), S. 1790–1793. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2003.
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  @article{Sauer2003, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Sauer, M}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {sauer}, number = 16, pages = {1790-1793}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer}, type = {Article}, volume = 42, year = 2003 }

  %0 Journal Article %1 Sauer2003 %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2003 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 16 %P 1790-1793 %T Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer %U http://dx.doi.org/10.1002/anie.200201611 %V 42
• Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores. Vosch, Tom; Cotlet, Mircea; Hofkens, Johan; Biest, Koen Van Der; Lor, Marc; Weston, Kenneth; Tinnefeld, Philip; Sauer, Markus; Latterini, Loredana; Müllen, Klaus; Schryver, Frans C. De. In The Journal of Physical Chemistry A, 107(36), S. 6920–6931. 2003.
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  Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system

  @article{doi:10.1021/jp034906d, abstract = {Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system}, author = {Vosch, Tom and Cotlet, Mircea and Hofkens, Johan and Biest, Koen Van Der and Lor, Marc and Weston, Kenneth and Tinnefeld, Philip and Sauer, Markus and Latterini, Loredana and Müllen, Klaus and Schryver, Frans C. De}, journal = {The Journal of Physical Chemistry A}, keywords = {sauer}, number = 36, pages = {6920-6931}, title = {Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores}, volume = 107, year = 2003 }

  %0 Journal Article %1 doi:10.1021/jp034906d %A Vosch, Tom %A Cotlet, Mircea %A Hofkens, Johan %A Biest, Koen Van Der %A Lor, Marc %A Weston, Kenneth %A Tinnefeld, Philip %A Sauer, Markus %A Latterini, Loredana %A Müllen, Klaus %A Schryver, Frans C. De %D 2003 %J The Journal of Physical Chemistry A %N 36 %P 6920-6931 %R 10.1021/jp034906d %T Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores %U http://pubs.acs.org/doi/abs/10.1021/jp034906d %V 107 %X Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system
• From linear genome sequence to three-dimensional organization of the cell nucleus. Politz, Joan; van Driel, Roel; Sauer, Markus; Pombo, Ana. In Genome Biology, 4(3), S. 1–4. BioMed Central, 2003.
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  A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.

  @article{springerlink:10.1186/gb-2003-4-3-310, abstract = {A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.}, author = {Politz, Joan and van Driel, Roel and Sauer, Markus and Pombo, Ana}, journal = {Genome Biology}, keywords = {sauer}, note = {10.1186/gb-2003-4-3-310}, pages = {1-4}, publisher = {BioMed Central}, title = {From linear genome sequence to three-dimensional organization of the cell nucleus}, volume = 4, year = 2003 }

  %0 Journal Article %1 springerlink:10.1186/gb-2003-4-3-310 %A Politz, Joan %A van Driel, Roel %A Sauer, Markus %A Pombo, Ana %D 2003 %I BioMed Central %J Genome Biology %P 1-4 %T From linear genome sequence to three-dimensional organization of the cell nucleus %U http://dx.doi.org/10.1186/gb-2003-4-3-310 %V 4 %X A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.
• Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching. Vaiana, Andrea C.; Schulz, Andreas; Wolfrum, Jürgen; Sauer, Markus; Smith, Jeremy C. In Journal of Computational Chemistry, 24(5), S. 632–639. Wiley Subscription Services, Inc., A Wiley Company, 2003.
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  Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003

  @article{JCC:JCC10190, abstract = {Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003}, author = {Vaiana, Andrea C. and Schulz, Andreas and Wolfrum, Jürgen and Sauer, Markus and Smith, Jeremy C.}, journal = {Journal of Computational Chemistry}, keywords = {sauer}, number = 5, pages = {632–639}, publisher = {Wiley Subscription Services, Inc., A Wiley Company}, title = {Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching}, volume = 24, year = 2003 }

  %0 Journal Article %1 JCC:JCC10190 %A Vaiana, Andrea C. %A Schulz, Andreas %A Wolfrum, Jürgen %A Sauer, Markus %A Smith, Jeremy C. %D 2003 %I Wiley Subscription Services, Inc., A Wiley Company %J Journal of Computational Chemistry %N 5 %P 632--639 %R 10.1002/jcc.10190 %T Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching %U http://dx.doi.org/10.1002/jcc.10190 %V 24 %X Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003
• Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Buschmann, V; Weston, KD; Sauer, M. In BIOCONJUGATE CHEMISTRY, 14(1), S. 195–204. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

  @article{Buschmann2003, abstract = {The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Buschmann, V and Weston, KD and Sauer, M}, journal = {BIOCONJUGATE CHEMISTRY}, keywords = {sauer}, month = {01}, number = 1, pages = {195-204}, publisher = {AMER CHEMICAL SOC}, title = {Spectroscopic study and evaluation of red-absorbing fluorescent dyes}, type = {Article}, volume = 14, year = 2003 }

  %0 Journal Article %1 Buschmann2003 %A Buschmann, V %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J BIOCONJUGATE CHEMISTRY %N 1 %P 195-204 %T Spectroscopic study and evaluation of red-absorbing fluorescent dyes %U http://dx.doi.org/10.1021/bc025600x %V 14 %X The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.
• Excitonic behavior of rhodamine dimers: A single-molecule study. Hernando, J; van der Schaaf, M; van Dijk, EMHP; Sauer, M; Garcia-Parajo, MF; van Hulst, NF. In JOURNAL OF PHYSICAL CHEMISTRY A, 107(1), S. 43–52. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.

  @article{Hernando2003, abstract = {The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Hernando, J and van der Schaaf, M and van Dijk, EMHP and Sauer, M and Garcia-Parajo, MF and van Hulst, NF}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {01}, number = 1, pages = {43-52}, publisher = {AMER CHEMICAL SOC}, title = {Excitonic behavior of rhodamine dimers: A single-molecule study}, type = {Article}, volume = 107, year = 2003 }

  %0 Journal Article %1 Hernando2003 %A Hernando, J %A van der Schaaf, M %A van Dijk, EMHP %A Sauer, M %A Garcia-Parajo, MF %A van Hulst, NF %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 1 %P 43-52 %T Excitonic behavior of rhodamine dimers: A single-molecule study %U http://dx.doi.org/10.1021/jp0218995 %V 107 %X The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.
• Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level. Neuweiler, H; Schulz, A; Bohmer, M; Enderlein, J; Sauer, M. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(18), S. 5324–5330. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

  @article{Neuweiler2003, abstract = {We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Schulz, A and Bohmer, M and Enderlein, J and Sauer, M}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {05}, number = 18, pages = {5324-5330}, publisher = {AMER CHEMICAL SOC}, title = {Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Neuweiler2003 %A Neuweiler, H %A Schulz, A %A Bohmer, M %A Enderlein, J %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 18 %P 5324-5330 %T Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level %U http://dx.doi.org/10.1021/ja034040p %V 125 %X We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.
• Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy. Knemeyer, JP; Herten, DP; Sauer, M. In ANALYTICAL CHEMISTRY, 75(9), S. 2147–2153. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.

  @article{Knemeyer2003, abstract = {The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30\% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Knemeyer, JP and Herten, DP and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = {05}, number = 9, pages = {2147-2153}, publisher = {AMER CHEMICAL SOC}, title = {Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy}, type = {Article}, volume = 75, year = 2003 }

  %0 Journal Article %1 Knemeyer2003 %A Knemeyer, JP %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 9 %P 2147-2153 %T Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1021/ac026333r %V 75 %X The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.
• A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer. Piestert, O; Barsch, H; Buschmann, V; Heinlein, T; Knemeyer, JP; Weston, KD; Sauer, M. In NANO LETTERS, 3(7), S. 979–982. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.

  @article{Piestert2003, abstract = {We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Piestert, O and Barsch, H and Buschmann, V and Heinlein, T and Knemeyer, JP and Weston, KD and Sauer, M}, journal = {NANO LETTERS}, keywords = {sauer}, month = {07}, number = 7, pages = {979-982}, publisher = {AMER CHEMICAL SOC}, title = {A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer}, type = {Article}, volume = 3, year = 2003 }

  %0 Journal Article %1 Piestert2003 %A Piestert, O %A Barsch, H %A Buschmann, V %A Heinlein, T %A Knemeyer, JP %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J NANO LETTERS %N 7 %P 979-982 %T A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer %U http://dx.doi.org/10.1021/nl0341988 %V 3 %X We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.
• Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins. Heinlein, T; Knemeyer, JP; Piestert, O; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY B, 107(31), S. 7957–7964. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.

  @article{Heinlein2003, abstract = {Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heinlein, T and Knemeyer, JP and Piestert, O and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = {08}, number = 31, pages = {7957-7964}, publisher = {AMER CHEMICAL SOC}, title = {Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins}, type = {Article}, volume = 107, year = 2003 }

  %0 Journal Article %1 Heinlein2003 %A Heinlein, T %A Knemeyer, JP %A Piestert, O %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 31 %P 7957-7964 %T Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins %U http://dx.doi.org/10.1021/jp0348068 %V 107 %X Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.
• Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules. Hofkens, J; Cotlet, M; Vosch, T; Tinnefeld, P; Weston, KD; Ego, C; Grimsdale, A; Mullen, K; Beljonne, D; Bredas, JL; Jordens, S; Schweitzer, G; Sauer, M; Schryver, F De. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(23), S. 13146–13151. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2003.
  • [ Abstract ]
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  We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.

  @article{Hofkens2003, abstract = {We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Hofkens, J and Cotlet, M and Vosch, T and Tinnefeld, P and Weston, KD and Ego, C and Grimsdale, A and Mullen, K and Beljonne, D and Bredas, JL and Jordens, S and Schweitzer, G and Sauer, M and Schryver, F De}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {sauer}, month = 11, number = 23, pages = {13146-13151}, publisher = {NATL ACAD SCIENCES}, title = {Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules}, type = {Article}, volume = 100, year = 2003 }

  %0 Journal Article %1 Hofkens2003 %A Hofkens, J %A Cotlet, M %A Vosch, T %A Tinnefeld, P %A Weston, KD %A Ego, C %A Grimsdale, A %A Mullen, K %A Beljonne, D %A Bredas, JL %A Jordens, S %A Schweitzer, G %A Sauer, M %A Schryver, F De %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2003 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 23 %P 13146-13151 %T Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules %U http://dx.doi.org/10.1073/pnas.2235805100 %V 100 %X We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.
• Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation. Vaiana, AC; Neuweiler, H; Schulz, A; Wolfrum, J; Sauer, M; Smith, JC. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(47), S. 14564–14572. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.

  @article{Vaiana2003a, abstract = {Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25\% of the R6G dye is fluorescent in comparison with 10\% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Vaiana, AC and Neuweiler, H and Schulz, A and Wolfrum, J and Sauer, M and Smith, JC}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = 11, number = 47, pages = {14564-14572}, publisher = {AMER CHEMICAL SOC}, title = {Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Vaiana2003a %A Vaiana, AC %A Neuweiler, H %A Schulz, A %A Wolfrum, J %A Sauer, M %A Smith, JC %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 47 %P 14564-14572 %T Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation %U http://dx.doi.org/10.1021/ja036082j %V 125 %X Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.
• Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Marme, N; Knemeyer, JP; Sauer, M; Wolfrum, J. In BIOCONJUGATE CHEMISTRY, 14(6), S. 1133–1139. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.

  @article{Marme2003, abstract = {Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Marme, N and Knemeyer, JP and Sauer, M and Wolfrum, J}, journal = {BIOCONJUGATE CHEMISTRY}, keywords = {sauer}, month = 11, number = 6, pages = {1133-1139}, publisher = {AMER CHEMICAL SOC}, title = {Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan}, type = {Article}, volume = 14, year = 2003 }

  %0 Journal Article %1 Marme2003 %A Marme, N %A Knemeyer, JP %A Sauer, M %A Wolfrum, J %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J BIOCONJUGATE CHEMISTRY %N 6 %P 1133-1139 %T Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan %U http://dx.doi.org/10.1021/bc0341324 %V 14 %X Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.
• Direct observation of collective blinking and energy transfer in a bichromophoric system. Tinnefeld, P; Buschmann, V; Weston, KD; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY A, 107(3), S. 323–327. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 --> T-N --> S-1 --> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.

  @article{Tinnefeld2003, abstract = {A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 –> T-N –> S-1 –> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tinnefeld, P and Buschmann, V and Weston, KD and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {01}, number = 3, pages = {323-327}, publisher = {AMER CHEMICAL SOC}, title = {Direct observation of collective blinking and energy transfer in a bichromophoric system}, type = {Letter}, volume = 107, year = 2003 }

  %0 Journal Article %1 Tinnefeld2003 %A Tinnefeld, P %A Buschmann, V %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 3 %P 323-327 %T Direct observation of collective blinking and energy transfer in a bichromophoric system %U http://dx.doi.org/10.1021/jp026565u %V 107 %X A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 --> T-N --> S-1 --> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.

• Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons. Weston, KD; Dyck, M; Tinnefeld, P; Muller, C; Herten, DP; Sauer, M. In ANALYTICAL CHEMISTRY, 74(20), S. 5342–5349. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2002.
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  A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

  @article{Weston2002, abstract = {A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Weston, KD and Dyck, M and Tinnefeld, P and Muller, C and Herten, DP and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 10, number = 20, pages = {5342-5349}, publisher = {AMER CHEMICAL SOC}, title = {Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons}, type = {Article}, volume = 74, year = 2002 }

  %0 Journal Article %1 Weston2002 %A Weston, KD %A Dyck, M %A Tinnefeld, P %A Muller, C %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2002 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 20 %P 5342-5349 %T Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons %U http://dx.doi.org/10.1021/ac025730z %V 74 %X A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.
• High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Heilemann, M; Herten, DP; Heintzmann, R; Cremer, C; Muller, C; Tinnefeld, P; Weston, KD; Wolfrum, J; Sauer, M. In ANALYTICAL CHEMISTRY, 74(14), S. 3511–3517. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2002.
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  Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.

  @article{Heilemann2002, abstract = {Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, M and Herten, DP and Heintzmann, R and Cremer, C and Muller, C and Tinnefeld, P and Weston, KD and Wolfrum, J and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {mike}, month = {07}, number = 14, pages = {3511-3517}, publisher = {AMER CHEMICAL SOC}, title = {High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy}, type = {Article}, volume = 74, year = 2002 }

  %0 Journal Article %1 Heilemann2002 %A Heilemann, M %A Herten, DP %A Heintzmann, R %A Cremer, C %A Muller, C %A Tinnefeld, P %A Weston, KD %A Wolfrum, J %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2002 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 14 %P 3511-3517 %T High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1021/ac025576g %V 74 %X Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.
• Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data. Dietrich, Anja; Buschmann, Volker; Müller, Christian; Sauer, Markus. In Reviews in Molecular Biotechnology, 82(3), S. 211–231. 2002.
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  We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

  @article{Dietrich2002211, abstract = {We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.}, author = {Dietrich, Anja and Buschmann, Volker and Müller, Christian and Sauer, Markus}, journal = {Reviews in Molecular Biotechnology}, keywords = {sauer}, number = 3, pages = {211 - 231}, title = {Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data}, volume = 82, year = 2002 }

  %0 Journal Article %1 Dietrich2002211 %A Dietrich, Anja %A Buschmann, Volker %A Müller, Christian %A Sauer, Markus %D 2002 %J Reviews in Molecular Biotechnology %N 3 %P 211 - 231 %R DOI: 10.1016/S1389-0352(01)00039-3 %T Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data %U http://www.sciencedirect.com/science/article/B6VR0-44VVW8G-4/2/5fe48e1df0969426c1254afbc5bb192b %V 82 %X We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.
• Cellular Uptake Studies with beta-Peptides. Rueping, Magnus; Mahajan, Yogesh; Sauer, Markus; Seebach, Dieter. In ChemBioChem, 3(2-3), S. 257–259. WILEY-VCH Verlag GmbH, 2002.
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  Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed

  @article{CBIC:CBIC257, abstract = {Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed}, author = {Rueping, Magnus and Mahajan, Yogesh and Sauer, Markus and Seebach, Dieter}, journal = {ChemBioChem}, keywords = {sauer}, number = {2-3}, pages = {257–259}, publisher = {WILEY-VCH Verlag GmbH}, title = {Cellular Uptake Studies with beta-Peptides}, volume = 3, year = 2002 }

  %0 Journal Article %1 CBIC:CBIC257 %A Rueping, Magnus %A Mahajan, Yogesh %A Sauer, Markus %A Seebach, Dieter %D 2002 %I WILEY-VCH Verlag GmbH %J ChemBioChem %N 2-3 %P 257--259 %R 10.1002/1439-7633(20020301)3:2/3<257::AID-CBIC257>3.0.CO;2-S %T Cellular Uptake Studies with beta-Peptides %U http://dx.doi.org/10.1002/1439-7633(20020301)3:2/3<257::AID-CBIC257>3.0.CO;2-S %V 3 %X Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed
• Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes. Neuweiler, Hannes; Schulz, Andreas; Vaiana, Andrea C.; Smith, Jeremy C.; Kaul, Sepp; Wolfrum, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, 41(24), S. 4769–4773. WILEY-VCH Verlag, 2002.
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  Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.

  @article{ANIE:ANIE200290044, abstract = {Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.}, author = {Neuweiler, Hannes and Schulz, Andreas and Vaiana, Andrea C. and Smith, Jeremy C. and Kaul, Sepp and Wolfrum, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {hannes}, number = 24, pages = {4769–4773}, publisher = {WILEY-VCH Verlag}, title = {Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes}, volume = 41, year = 2002 }

  %0 Journal Article %1 ANIE:ANIE200290044 %A Neuweiler, Hannes %A Schulz, Andreas %A Vaiana, Andrea C. %A Smith, Jeremy C. %A Kaul, Sepp %A Wolfrum, Jürgen %A Sauer, Markus %D 2002 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 24 %P 4769--4773 %R 10.1002/anie.200290044 %T Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes %U http://dx.doi.org/10.1002/anie.200290044 %V 41 %X Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.
• Antibunching in the Emission of a Single Tetrachromophoric Dendritic System. Tinnefeld, Philip; Weston, Kenneth D.; Vosch, Tom; Cotlet, Mircea; Weil, Tanja; Hofkens, Johan; Müllen, Klaus; Schryver, Frans C. De; Sauer, Markus. In Journal of the American Chemical Society, 124(48), S. 14310–14311. 2002.
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  The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

  @article{doi:10.1021/ja027343c, abstract = {The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.}, author = {Tinnefeld, Philip and Weston, Kenneth D. and Vosch, Tom and Cotlet, Mircea and Weil, Tanja and Hofkens, Johan and Müllen, Klaus and Schryver, Frans C. De and Sauer, Markus}, journal = {Journal of the American Chemical Society}, keywords = {sauer}, number = 48, pages = {14310-14311}, title = {Antibunching in the Emission of a Single Tetrachromophoric Dendritic System}, volume = 124, year = 2002 }

  %0 Journal Article %1 doi:10.1021/ja027343c %A Tinnefeld, Philip %A Weston, Kenneth D. %A Vosch, Tom %A Cotlet, Mircea %A Weil, Tanja %A Hofkens, Johan %A Müllen, Klaus %A Schryver, Frans C. De %A Sauer, Markus %D 2002 %J Journal of the American Chemical Society %N 48 %P 14310-14311 %R 10.1021/ja027343c %T Antibunching in the Emission of a Single Tetrachromophoric Dendritic System %U http://pubs.acs.org/doi/abs/10.1021/ja027343c %V 124 %X The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

• Time-varying photon probability distribution of individual molecules at room temperature. Tinnefeld, P.; Müller, C.; Sauer, M. In Chemical Physics Letters, 345(3-4), S. 252–258. 2001.
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  The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time

  @article{Tinnefeld2001252, abstract = {The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time}, author = {Tinnefeld, P. and Müller, C. and Sauer, M.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {252 - 258}, title = {Time-varying photon probability distribution of individual molecules at room temperature}, volume = 345, year = 2001 }

  %0 Journal Article %1 Tinnefeld2001252 %A Tinnefeld, P. %A Müller, C. %A Sauer, M. %D 2001 %J Chemical Physics Letters %N 3-4 %P 252 - 258 %R DOI: 10.1016/S0009-2614(01)00883-1 %T Time-varying photon probability distribution of individual molecules at room temperature %U http://www.sciencedirect.com/science/article/B6TFN-43XFWP4-7/2/9500b916e6f748d7c43dbff85a2e3e4f %V 345 %X The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time
• Electron induced chemical nanolithography with self-assembled monolayers. Geyer, W.; Stadler, V.; Eck, W.; Golzhauser, A.; Grunze, M.; Sauer, M.; Weimann, T.; Hinze, P. In The 45th international conference on electron, ion, and photon beam technology and nanofabrication, Bd. 19, S. 2732–2735. AVS, Washington, DC (USA), 2001.
  • [ Abstract ]
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  We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society

  @conference{geyer:2732, abstract = {We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society}, author = {Geyer, W. and Stadler, V. and Eck, W. and Golzhauser, A. and Grunze, M. and Sauer, M. and Weimann, T. and Hinze, P.}, journal = {The 45th international conference on electron, ion, and photon beam technology and nanofabrication}, keywords = {sauer}, number = 6, pages = {2732-2735}, publisher = {AVS}, title = {Electron induced chemical nanolithography with self-assembled monolayers}, volume = 19, year = 2001 }

  %0 Generic %1 geyer:2732 %A Geyer, W. %A Stadler, V. %A Eck, W. %A Golzhauser, A. %A Grunze, M. %A Sauer, M. %A Weimann, T. %A Hinze, P. %D 2001 %I AVS %J The 45th international conference on electron, ion, and photon beam technology and nanofabrication %N 6 %P 2732-2735 %R 10.1116/1.1421560 %T Electron induced chemical nanolithography with self-assembled monolayers %U http://link.aip.org/link/?JVB/19/2732/1 %V 19 %X We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society
• Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting. Enderlein, Jörg; Sauer, Markus. In The Journal of Physical Chemistry A, 105(1), S. 48–53. 2001.
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  A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data

  @article{doi:10.1021/jp002358n, abstract = {A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data}, author = {Enderlein, Jörg and Sauer, Markus}, journal = {The Journal of Physical Chemistry A}, keywords = {sauer}, number = 1, pages = {48-53}, title = {Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting}, volume = 105, year = 2001 }

  %0 Journal Article %1 doi:10.1021/jp002358n %A Enderlein, Jörg %A Sauer, Markus %D 2001 %J The Journal of Physical Chemistry A %N 1 %P 48-53 %R 10.1021/jp002358n %T Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting %U http://pubs.acs.org/doi/abs/10.1021/jp002358n %V 105 %X A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data
• Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects. Sauer, M.; Angerer, B.; Ankenbauer, W.; Földes-Papp, Z.; Göbel, F.; Han, K.-T.; Rigler, R.; Schulz, A.; Wolfrum, J.; Zander, C. In Journal of Biotechnology, 86(3), S. 181–201. 2001.
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  We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed ~50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

  @article{Sauer2001181, abstract = {We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed  50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.}, author = {Sauer, M. and Angerer, B. and Ankenbauer, W. and Földes-Papp, Z. and Göbel, F. and Han, K.-T. and Rigler, R. and Schulz, A. and Wolfrum, J. and Zander, C.}, journal = {Journal of Biotechnology}, keywords = {sauer}, note = {DNA-Sequencing at the Single Molecule Level}, number = 3, pages = {181 - 201}, title = {Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects}, volume = 86, year = 2001 }

  %0 Journal Article %1 Sauer2001181 %A Sauer, M. %A Angerer, B. %A Ankenbauer, W. %A Földes-Papp, Z. %A Göbel, F. %A Han, K.-T. %A Rigler, R. %A Schulz, A. %A Wolfrum, J. %A Zander, C. %D 2001 %J Journal of Biotechnology %N 3 %P 181 - 201 %R DOI: 10.1016/S0168-1656(00)00413-2 %T Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects %U http://www.sciencedirect.com/science/article/B6T3C-42JHD6H-3/2/8ed6101d2ec2e07a4cf2411397f9a399 %V 86 %X We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed ~50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.
• Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM). Tinnefeld, P; Herten, DP; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY A, 105(34), S. 7989–8003. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2001.
  • [ Abstract ]
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  A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.

  @article{Tinnefeld2001, abstract = {A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15\% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tinnefeld, P and Herten, DP and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {08}, number = 34, pages = {7989-8003}, publisher = {AMER CHEMICAL SOC}, title = {Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM)}, type = {Article}, volume = 105, year = 2001 }

  %0 Journal Article %1 Tinnefeld2001 %A Tinnefeld, P %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2001 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 34 %P 7989-8003 %T Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) %U http://dx.doi.org/10.1021/jp010365l %V 105 %X A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.
• On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives. Rueping, M; Dietrich, A; Buschmann, V; Fritz, MG; Sauer, M; Seebach, D. In MACROMOLECULES, 34(20), S. 7042–7048. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2001.
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  Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.

  @article{Rueping2001, abstract = {Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Rueping, M and Dietrich, A and Buschmann, V and Fritz, MG and Sauer, M and Seebach, D}, journal = {MACROMOLECULES}, keywords = {sauer}, month = {09}, number = 20, pages = {7042-7048}, publisher = {AMER CHEMICAL SOC}, title = {On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives}, type = {Article}, volume = 34, year = 2001 }

  %0 Journal Article %1 Rueping2001 %A Rueping, M %A Dietrich, A %A Buschmann, V %A Fritz, MG %A Sauer, M %A Seebach, D %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2001 %I AMER CHEMICAL SOC %J MACROMOLECULES %N 20 %P 7042-7048 %T On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives %U http://dx.doi.org/10.1021/ma010520u %V 34 %X Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.
• Time-gated biological imaging by use of colloidal quantum dots. Dahan, M.; Laurence, T.; Pinaud, F.; Chemla, D. S.; Alivisatos, A. P.; Sauer, M.; Weiss, S. In Opt. Lett., 26(11), S. 825–827. OSA, 2001.
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  The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.

  @article{Dahan:01, abstract = {The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.}, author = {Dahan, M. and Laurence, T. and Pinaud, F. and Chemla, D. S. and Alivisatos, A. P. and Sauer, M. and Weiss, S.}, journal = {Opt. Lett.}, keywords = {sauer}, month = {06}, number = 11, pages = {825–827}, publisher = {OSA}, title = {Time-gated biological imaging by use of colloidal quantum dots}, volume = 26, year = 2001 }

  %0 Journal Article %1 Dahan:01 %A Dahan, M. %A Laurence, T. %A Pinaud, F. %A Chemla, D. S. %A Alivisatos, A. P. %A Sauer, M. %A Weiss, S. %D 2001 %I OSA %J Opt. Lett. %N 11 %P 825--827 %R 10.1364/OL.26.000825 %T Time-gated biological imaging by use of colloidal quantum dots %U http://ol.osa.org/abstract.cfm?URI=ol-26-11-825 %V 26 %X The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.

• Probes for Detection of Specific DNA Sequences at the Single-Molecule Level. Knemeyer, Jens-Peter; Marmé, Nicole; Sauer, Markus. In Analytical Chemistry, 72(16), S. 3717–3724. 2000.
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  A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules

  @article{doi:10.1021/ac000024o, abstract = {A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules}, author = {Knemeyer, Jens-Peter and Marmé, Nicole and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 16, pages = {3717-3724}, title = {Probes for Detection of Specific DNA Sequences at the Single-Molecule Level}, volume = 72, year = 2000 }

  %0 Journal Article %1 doi:10.1021/ac000024o %A Knemeyer, Jens-Peter %A Marmé, Nicole %A Sauer, Markus %D 2000 %J Analytical Chemistry %N 16 %P 3717-3724 %R 10.1021/ac000024o %T Probes for Detection of Specific DNA Sequences at the Single-Molecule Level %U http://pubs.acs.org/doi/abs/10.1021/ac000024o %V 72 %X A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules
• Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting. Herten, D.P.; Tinnefeld, P.; Sauer, M. In Applied Physics B: Lasers and Optics, 71(5), S. 765–771. Springer Berlin / Heidelberg, 2000.
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  We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.

  @article{springerlink:10.1007/s003400000405, abstract = {We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.}, author = {Herten, D.P. and Tinnefeld, P. and Sauer, M.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400000405}, pages = {765-771}, publisher = {Springer Berlin / Heidelberg}, title = {Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting}, volume = 71, year = 2000 }

  %0 Journal Article %1 springerlink:10.1007/s003400000405 %A Herten, D.P. %A Tinnefeld, P. %A Sauer, M. %D 2000 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 765-771 %T Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting %U http://dx.doi.org/10.1007/s003400000405 %V 71 %X We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.
• 7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain. Seela, Frank; Zulauf, Matthias; Sauer, Markus; Deimel, Michael. In Helvetica Chimica Acta, 83(5), S. 910–927. Verlag Helvetica Chimica Acta, 2000.
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  7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).

  @article{HLCA:HLCA910, abstract = {7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).}, author = {Seela, Frank and Zulauf, Matthias and Sauer, Markus and Deimel, Michael}, journal = {Helvetica Chimica Acta}, keywords = {sauer}, number = 5, pages = {910–927}, publisher = {Verlag Helvetica Chimica Acta}, title = {7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain}, volume = 83, year = 2000 }

  %0 Journal Article %1 HLCA:HLCA910 %A Seela, Frank %A Zulauf, Matthias %A Sauer, Markus %A Deimel, Michael %D 2000 %I Verlag Helvetica Chimica Acta %J Helvetica Chimica Acta %N 5 %P 910--927 %R 10.1002/(SICI)1522-2675(20000510)83:5<910::AID-HLCA910>3.0.CO;2-4 %T 7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain %U http://dx.doi.org/10.1002/(SICI)1522-2675(20000510)83:5<910::AID-HLCA910>3.0.CO;2-4 %V 83 %X 7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).
• Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments. Neumann, M.; Herten, D.-P.; Dietrich, A.; Wolfrum, J.; Sauer, M. In Journal of Chromatography A, 871(1-2), S. 299–310. 2000.
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  The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

  @article{Neumann2000299, abstract = {The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.}, author = {Neumann, M. and Herten, D.-P. and Dietrich, A. and Wolfrum, J. and Sauer, M.}, journal = {Journal of Chromatography A}, keywords = {sauer}, number = {1-2}, pages = {299 - 310}, title = {Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments}, volume = 871, year = 2000 }

  %0 Journal Article %1 Neumann2000299 %A Neumann, M. %A Herten, D.-P. %A Dietrich, A. %A Wolfrum, J. %A Sauer, M. %D 2000 %J Journal of Chromatography A %N 1-2 %P 299 - 310 %R DOI: 10.1016/S0021-9673(99)00909-7 %T Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments %U http://www.sciencedirect.com/science/article/B6TG8-3YMF6PM-14/2/5e863581c92280fc7d00c7225fc50baa %V 871 %X The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.
• Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes. Irngartinger, Hermann; Fettel, Peter Walter; Escher, Thomas; Tinnefeld, Philip; Nord, Simon; Sauer, Markus. In European Journal of Organic Chemistry, 2000(3), S. 455–465. WILEY-VCH Verlag GmbH, 2000.
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  The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.

  @article{EJOC:EJOC455, abstract = {The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.}, author = {Irngartinger, Hermann and Fettel, Peter Walter and Escher, Thomas and Tinnefeld, Philip and Nord, Simon and Sauer, Markus}, journal = {European Journal of Organic Chemistry}, keywords = {sauer}, number = 3, pages = {455–465}, publisher = {WILEY-VCH Verlag GmbH}, title = {Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes}, volume = 2000, year = 2000 }

  %0 Journal Article %1 EJOC:EJOC455 %A Irngartinger, Hermann %A Fettel, Peter Walter %A Escher, Thomas %A Tinnefeld, Philip %A Nord, Simon %A Sauer, Markus %D 2000 %I WILEY-VCH Verlag GmbH %J European Journal of Organic Chemistry %N 3 %P 455--465 %R 10.1002/(SICI)1099-0690(200002)2000:3<455::AID-EJOC455>3.0.CO;2-N %T Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes %U http://dx.doi.org/10.1002/(SICI)1099-0690(200002)2000:3<455::AID-EJOC455>3.0.CO;2-N %V 2000 %X The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.
• Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level. Tinnefeld, Philip; Buschmann, Volker; Herten, Dirk-Peter; Han, Kyung-Tae; Sauer, Markus. In Single Molecules, 1(3), S. 215–223. WILEY-VCH Verlag Berlin GmbH, 2000.
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  We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

  @article{SIMO:SIMO215, abstract = {We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of  0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.}, author = {Tinnefeld, Philip and Buschmann, Volker and Herten, Dirk-Peter and Han, Kyung-Tae and Sauer, Markus}, journal = {Single Molecules}, keywords = {sauer}, number = 3, pages = {215–223}, publisher = {WILEY-VCH Verlag Berlin GmbH}, title = {Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level}, volume = 1, year = 2000 }

  %0 Journal Article %1 SIMO:SIMO215 %A Tinnefeld, Philip %A Buschmann, Volker %A Herten, Dirk-Peter %A Han, Kyung-Tae %A Sauer, Markus %D 2000 %I WILEY-VCH Verlag Berlin GmbH %J Single Molecules %N 3 %P 215--223 %R 10.1002/1438-5171(200009)1:3<215::AID-SIMO215>3.0.CO;2-S %T Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level %U http://dx.doi.org/10.1002/1438-5171(200009)1:3<215::AID-SIMO215>3.0.CO;2-S %V 1 %X We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

• Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures. Neumann, M.; Ehrfeld, W.; Han, K.-T.; Jacob, P.; Konrad, R.; Siebert, S.; Wolfrum, J.; Sauer, M. In Biomedical Chromatography, 13(2), S. 109–110. John Wiley & Sons, Ltd., 1999.
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  @article{BMC:BMC854, author = {Neumann, M. and Ehrfeld, W. and Han, K.-T. and Jacob, P. and Konrad, R. and Siebert, S. and Wolfrum, J. and Sauer, M.}, journal = {Biomedical Chromatography}, keywords = {sauer}, number = 2, pages = {109–110}, publisher = {John Wiley & Sons, Ltd.}, title = {Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures}, volume = 13, year = 1999 }

  %0 Journal Article %1 BMC:BMC854 %A Neumann, M. %A Ehrfeld, W. %A Han, K.-T. %A Jacob, P. %A Konrad, R. %A Siebert, S. %A Wolfrum, J. %A Sauer, M. %D 1999 %I John Wiley & Sons, Ltd. %J Biomedical Chromatography %N 2 %P 109--110 %R 10.1002/(SICI)1099-0801(199904)13:2<109::AID-BMC854>3.0.CO;2-R %T Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures %U http://dx.doi.org/10.1002/(SICI)1099-0801(199904)13:2<109::AID-BMC854>3.0.CO;2-R %V 13
• Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC). Becker, W.; Hickl, H.; Zander, C.; Drexhage, K. H.; Sauer, M.; Siebert, S.; Wolfrum, J. In Review of Scientific Instruments, 70(3), S. 1835–1841. American Institute of Physics, 1999.
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  A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.

  @article{becker1999timeresolved, abstract = {A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.}, author = {Becker, W. and Hickl, H. and Zander, C. and Drexhage, K. H. and Sauer, M. and Siebert, S. and Wolfrum, J.}, booktitle = {Review of Scientific Instruments}, journal = {Review of Scientific Instruments}, keywords = {sauer}, month = {03}, number = 3, pages = {1835–1841}, publisher = {American Institute of Physics}, title = {Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)}, volume = 70, year = 1999 }

  %0 Journal Article %1 becker1999timeresolved %A Becker, W. %A Hickl, H. %A Zander, C. %A Drexhage, K. H. %A Sauer, M. %A Siebert, S. %A Wolfrum, J. %B Review of Scientific Instruments %D 1999 %I American Institute of Physics %J Review of Scientific Instruments %N 3 %P 1835--1841 %R 10.1063/1.1149677 %T Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC) %U https://doi.org/10.1063/1.1149677 %V 70 %X A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.
• Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique. Sauer, M; Angerer, B; Han, K-T; Zander, C. In Phys. Chem. Chem. Phys., 1(10), S. 2471–2477. The Royal Society of Chemistry, 1999.
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  We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.

  @article{sauer1999detection, abstract = {We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.}, author = {Sauer, M and Angerer, B and Han, K-T and Zander, C}, journal = {Phys. Chem. Chem. Phys.}, keywords = {sauer}, number = 10, pages = {2471–2477}, publisher = {The Royal Society of Chemistry}, title = {Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique}, volume = 1, year = 1999 }

  %0 Journal Article %1 sauer1999detection %A Sauer, M %A Angerer, B %A Han, K-T %A Zander, C %D 1999 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %N 10 %P 2471--2477 %R 10.1039/A901411J %T Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique %U http://dx.doi.org/10.1039/A901411J %V 1 %X We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.

• Single-molecule counting and identification in a microcapillary. Zander, C.; Drexhage, K.H.; Han, K.-T.; Wolfrum, J.; Sauer, M. In Chemical Physics Letters, 286(5-6), S. 457–465. 1998.
  • [ Abstract ]
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  Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.

  @article{Zander1998457, abstract = {Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.}, author = {Zander, C. and Drexhage, K.H. and Han, K.-T. and Wolfrum, J. and Sauer, M.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {5-6}, pages = {457 - 465}, title = {Single-molecule counting and identification in a microcapillary}, volume = 286, year = 1998 }

  %0 Journal Article %1 Zander1998457 %A Zander, C. %A Drexhage, K.H. %A Han, K.-T. %A Wolfrum, J. %A Sauer, M. %D 1998 %J Chemical Physics Letters %N 5-6 %P 457 - 465 %R DOI: 10.1016/S0009-2614(98)00096-7 %T Single-molecule counting and identification in a microcapillary %U http://www.sciencedirect.com/science/article/B6TFN-3VN2MVG-3B/2/6f1d57b094658a541d630753d82f717f %V 286 %X Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.
• Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection. Lieberwirth, Ulrike; Arden-Jacob, Jutta; Drexhage, Karl H.; Herten, Dirk P.; Müller, Ralph; Neumann, Michael; Schulz, Andreas; Siebert, Stefan; Sagner, Gregor; Klingel, Sven; Sauer, Markus; Wolfrum, Jürgen. In Analytical Chemistry, 70(22), S. 4771–4779. 1998.
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  A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods

  @article{doi:10.1021/ac980230k, abstract = {A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods}, author = {Lieberwirth, Ulrike and Arden-Jacob, Jutta and Drexhage, Karl H. and Herten, Dirk P. and Müller, Ralph and Neumann, Michael and Schulz, Andreas and Siebert, Stefan and Sagner, Gregor and Klingel, Sven and Sauer, Markus and Wolfrum, Jürgen}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 22, pages = {4771-4779}, title = {Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection}, volume = 70, year = 1998 }

  %0 Journal Article %1 doi:10.1021/ac980230k %A Lieberwirth, Ulrike %A Arden-Jacob, Jutta %A Drexhage, Karl H. %A Herten, Dirk P. %A Müller, Ralph %A Neumann, Michael %A Schulz, Andreas %A Siebert, Stefan %A Sagner, Gregor %A Klingel, Sven %A Sauer, Markus %A Wolfrum, Jürgen %D 1998 %J Analytical Chemistry %N 22 %P 4771-4779 %R 10.1021/ac980230k %T Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection %U http://pubs.acs.org/doi/abs/10.1021/ac980230k %V 70 %X A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods
• Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase. SEELA, F.; CHEN, Y.; SAUER, M. In ChemInform, 29(28), S. no-no. WILEY-VCH Verlag, 1998.
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  Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

  @article{CHIN:CHIN199828268, abstract = {Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.}, author = {SEELA, F. and CHEN, Y. and SAUER, M.}, journal = {ChemInform}, keywords = {sauer}, number = 28, pages = {no–no}, publisher = {WILEY-VCH Verlag}, title = {Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase.}, volume = 29, year = 1998 }

  %0 Journal Article %1 CHIN:CHIN199828268 %A SEELA, F. %A CHEN, Y. %A SAUER, M. %D 1998 %I WILEY-VCH Verlag %J ChemInform %N 28 %P no--no %R 10.1002/chin.199828268 %T Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase. %U http://dx.doi.org/10.1002/chin.199828268 %V 29 %X Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
• Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level. Sauer, M; Drexhage, K.H; Lieberwirth, U; Müller, R; Nord, S; Zander, C. In Chemical Physics Letters, 284(3-4), S. 153–163. 1998.
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  Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.

  @article{Sauer1998153, abstract = {Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.}, author = {Sauer, M and Drexhage, K.H and Lieberwirth, U and Müller, R and Nord, S and Zander, C}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {153 - 163}, title = {Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level}, volume = 284, year = 1998 }

  %0 Journal Article %1 Sauer1998153 %A Sauer, M %A Drexhage, K.H %A Lieberwirth, U %A Müller, R %A Nord, S %A Zander, C %D 1998 %J Chemical Physics Letters %N 3-4 %P 153 - 163 %R DOI: 10.1016/S0009-2614(97)01377-8 %T Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level %U http://www.sciencedirect.com/science/article/B6TFN-3VN2FFT-29/2/0d5cca7d9662f7bc3754f4f96d675166 %V 284 %X Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.
• Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers. Sauer, Markus; Arden-Jacob, Jutta; Drexhage, Karl H; Göbel, Florian; Lieberwirth, Ulrike; Mühlegger, Klaus; Müller, Ralph; Wolfrum, Jürgen; Zander, Christoph. In Bioimaging, 6(1), S. 14–24. IOP Publishing Ltd, 1998.
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  We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)

  @article{BIO:BIO3, abstract = {We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)}, author = {Sauer, Markus and Arden-Jacob, Jutta and Drexhage, Karl H and Göbel, Florian and Lieberwirth, Ulrike and Mühlegger, Klaus and Müller, Ralph and Wolfrum, Jürgen and Zander, Christoph}, journal = {Bioimaging}, keywords = {sauer}, number = 1, pages = {14–24}, publisher = {IOP Publishing Ltd}, title = {Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers}, volume = 6, year = 1998 }

  %0 Journal Article %1 BIO:BIO3 %A Sauer, Markus %A Arden-Jacob, Jutta %A Drexhage, Karl H %A Göbel, Florian %A Lieberwirth, Ulrike %A Mühlegger, Klaus %A Müller, Ralph %A Wolfrum, Jürgen %A Zander, Christoph %D 1998 %I IOP Publishing Ltd %J Bioimaging %N 1 %P 14--24 %R 10.1002/1361-6374(199803)6:1<14::AID-BIO3>3.0.CO;2-O %T Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers %U http://dx.doi.org/10.1002/1361-6374(199803)6:1<14::AID-BIO3>3.0.CO;2-O %V 6 %X We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)

• Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes. Lieberwirth, U.; Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Nord, S.; Seeger, S.; Schulz, A; Wolfrum, J. In J. Fluoresc., 7, S. 55–58. 1997.
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  @article{lieberwirth1997development, author = {Lieberwirth, U. and Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Nord, S. and Seeger, S. and Schulz, A and Wolfrum, J.}, journal = {J. Fluoresc.}, keywords = {sauer}, pages = {55-58}, title = {Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes}, volume = 7, year = 1997 }

  %0 Journal Article %1 lieberwirth1997development %A Lieberwirth, U. %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Nord, S. %A Seeger, S. %A Schulz, A %A Wolfrum, J. %D 1997 %J J. Fluoresc. %P 55-58 %T Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes %V 7
• Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine. Nord, S.; Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Lieberwirth, U.; Seeger, S.; Wolfrum, J. In J. Fluoresc., 7, S. 15–18. 1997.
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  @article{nord1997ground, author = {Nord, S. and Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Lieberwirth, U. and Seeger, S. and Wolfrum, J.}, journal = {J. Fluoresc.}, keywords = {sauer}, pages = {15-18}, title = {Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine}, volume = 7, year = 1997 }

  %0 Journal Article %1 nord1997ground %A Nord, S. %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Lieberwirth, U. %A Seeger, S. %A Wolfrum, J. %D 1997 %J J. Fluoresc. %P 15-18 %T Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine %V 7
• Determination of the diffusion coefficient of dye in solution at single molecule level. Ko, D. S.; Sauer, M.; Nord, S.; Müller, R.; Wolfrum, J. In Chemical Physics Letters, 269(1-2), S. 54–58. 1997.
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  We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.

  @article{Ko199754, abstract = {We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.}, author = {Ko, D. S. and Sauer, M. and Nord, S. and Müller, R. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {1-2}, pages = {54 - 58}, title = {Determination of the diffusion coefficient of dye in solution at single molecule level}, volume = 269, year = 1997 }

  %0 Journal Article %1 Ko199754 %A Ko, D. S. %A Sauer, M. %A Nord, S. %A Müller, R. %A Wolfrum, J. %D 1997 %J Chemical Physics Letters %N 1-2 %P 54 - 58 %R DOI: 10.1016/S0009-2614(97)00246-7 %T Determination of the diffusion coefficient of dye in solution at single molecule level %U http://www.sciencedirect.com/science/article/B6TFN-3S9MRPW-4G/2/74f26e268928991c260b84a3f926321a %V 269 %X We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.
• Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set. Müller, Ralph; Herten, Dirk P.; Lieberwirth, Ulrike; Neumann, Michael; Sauer, Markus; Schulz, Andreas; Siebert, Stefan; Drexhage, Karl H.; Wolfrum, Jürgen. In Chemical Physics Letters, 279(5-6), S. 282–288. 1997.
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  A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.

  @article{Müller1997282, abstract = {A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.}, author = {Müller, Ralph and Herten, Dirk P. and Lieberwirth, Ulrike and Neumann, Michael and Sauer, Markus and Schulz, Andreas and Siebert, Stefan and Drexhage, Karl H. and Wolfrum, Jürgen}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {5-6}, pages = {282 - 288}, title = {Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set}, volume = 279, year = 1997 }

  %0 Journal Article %1 Müller1997282 %A Müller, Ralph %A Herten, Dirk P. %A Lieberwirth, Ulrike %A Neumann, Michael %A Sauer, Markus %A Schulz, Andreas %A Siebert, Stefan %A Drexhage, Karl H. %A Wolfrum, Jürgen %D 1997 %J Chemical Physics Letters %N 5-6 %P 282 - 288 %R DOI: 10.1016/S0009-2614(97)01041-5 %T Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set %U http://www.sciencedirect.com/science/article/B6TFN-3SFVBFC-47/2/985d40ed8deaa5d0ca07432857da5550 %V 279 %X A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.
• On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis. Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Marx, N. J.; Karger, A. E.; Lieberwirth, U.; Müller, R.; Neumann, M.; Nord, S.; Paulus, A.; Schulz, A.; Seeger, S.; Zander, C.; Wolfrum, J. In Biomedical Chromatography, 11(2), S. 81–82. John Wiley & Sons, Ltd., 1997.
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  @article{BMC:BMC645, author = {Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Marx, N. J. and Karger, A. E. and Lieberwirth, U. and Müller, R. and Neumann, M. and Nord, S. and Paulus, A. and Schulz, A. and Seeger, S. and Zander, C. and Wolfrum, J.}, journal = {Biomedical Chromatography}, keywords = {sauer}, number = 2, pages = {81–82}, publisher = {John Wiley & Sons, Ltd.}, title = {On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis}, volume = 11, year = 1997 }

  %0 Journal Article %1 BMC:BMC645 %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Marx, N. J. %A Karger, A. E. %A Lieberwirth, U. %A Müller, R. %A Neumann, M. %A Nord, S. %A Paulus, A. %A Schulz, A. %A Seeger, S. %A Zander, C. %A Wolfrum, J. %D 1997 %I John Wiley & Sons, Ltd. %J Biomedical Chromatography %N 2 %P 81--82 %R 10.1002/(SICI)1099-0801(199703)11:2<81::AID-BMC645>3.0.CO;2-U %T On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis %U http://dx.doi.org/10.1002/(SICI)1099-0801(199703)11:2<81::AID-BMC645>3.0.CO;2-U %V 11
• Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Sauer, M.; Zander, C.; Müller, R.; Ullrich, B.; Drexhage, K.H.; Kaul, S.; Wolfrum, J. In Applied Physics B: Lasers and Optics, 65(3), S. 427–431. Springer Berlin / Heidelberg, 1997.
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   M without separation steps.

  @article{springerlink:10.1007/s003400050292, abstract = { M without separation steps.}, author = {Sauer, M. and Zander, C. and Müller, R. and Ullrich, B. and Drexhage, K.H. and Kaul, S. and Wolfrum, J.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400050292}, pages = {427-431}, publisher = {Springer Berlin / Heidelberg}, title = {Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers}, volume = 65, year = 1997 }

  %0 Journal Article %1 springerlink:10.1007/s003400050292 %A Sauer, M. %A Zander, C. %A Müller, R. %A Ullrich, B. %A Drexhage, K.H. %A Kaul, S. %A Wolfrum, J. %D 1997 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 427-431 %T Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers %U http://dx.doi.org/10.1007/s003400050292 %V 65 %X  M without separation steps.

• Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies. Seidel, Claus A. M.; Schulz, Andreas; Sauer, Markus H. M. In The Journal of Physical Chemistry, 100(13), S. 5541–5553. 1996.
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  Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.

  @article{doi:10.1021/jp951507c, abstract = {Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.}, author = {Seidel, Claus A. M. and Schulz, Andreas and Sauer, Markus H. M.}, journal = {The Journal of Physical Chemistry}, keywords = {sauer}, number = 13, pages = {5541-5553}, title = {Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies}, volume = 100, year = 1996 }

  %0 Journal Article %1 doi:10.1021/jp951507c %A Seidel, Claus A. M. %A Schulz, Andreas %A Sauer, Markus H. M. %D 1996 %J The Journal of Physical Chemistry %N 13 %P 5541-5553 %R 10.1021/jp951507c %T Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies %U http://pubs.acs.org/doi/abs/10.1021/jp951507c %V 100 %X Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.
• Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser. Müller, R.; Zander, C.; Sauer, M.; Deimel, M.; Ko, D. S.; Siebert, S.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H.; Wolfrum, J. In Chemical Physics Letters, 262(6), S. 716–722. 1996.
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  We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).

  @article{Müller1996716, abstract = {We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).}, author = {Müller, R. and Zander, C. and Sauer, M. and Deimel, M. and Ko, D. S. and Siebert, S. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Drexhage, K. H. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = 6, pages = {716 - 722}, title = {Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser}, volume = 262, year = 1996 }

  %0 Journal Article %1 Müller1996716 %A Müller, R. %A Zander, C. %A Sauer, M. %A Deimel, M. %A Ko, D. S. %A Siebert, S. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Drexhage, K. H. %A Wolfrum, J. %D 1996 %J Chemical Physics Letters %N 6 %P 716 - 722 %R DOI: 10.1016/S0009-2614(96)01147-5 %T Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser %U http://www.sciencedirect.com/science/article/B6TFN-3VV60GR-2X/2/d00a127d7acc02c711f22e8392d20f98 %V 262 %X We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).
• Diode laser based detection of single molecules in solutions. Sauer, M.; Drexhage, K. H.; Zander, C.; Wolfrum, J. In Chemical Physics Letters, 254(3-4), S. 223–228. 1996.
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  Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.

  @article{Sauer1996223, abstract = {Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.}, author = {Sauer, M. and Drexhage, K. H. and Zander, C. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {223 - 228}, title = {Diode laser based detection of single molecules in solutions}, volume = 254, year = 1996 }

  %0 Journal Article %1 Sauer1996223 %A Sauer, M. %A Drexhage, K. H. %A Zander, C. %A Wolfrum, J. %D 1996 %J Chemical Physics Letters %N 3-4 %P 223 - 228 %R DOI: 10.1016/0009-2614(96)00303-X %T Diode laser based detection of single molecules in solutions %U http://www.sciencedirect.com/science/article/B6TFN-3V8YMT0-3K/2/b453bf51d51141737d98f04f0a321859 %V 254 %X Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.
• Detection and characterization of single molecules in aqueous solution. Zander, C.; Sauer, M.; Drexhage, K. H.; Ko, D.-S.; Schulz, A.; Wolfrum, J.; Brand, L.; Eggeling, C.; Seidel, C. A. M. In Applied Physics B: Lasers and Optics, 63(5), S. 517–523. Springer Berlin / Heidelberg, 1996.
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  Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100&#114ps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79&#450.33&#114ns (RB) and 3.79&#450.38&#114ns (R6G) in aqueous solution.

  @article{zander1996detection, abstract = {Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100rps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79ǂ.33rns (RB) and 3.79ǂ.38rns (R6G) in aqueous solution.}, author = {Zander, C. and Sauer, M. and Drexhage, K. H. and Ko, D.-S. and Schulz, A. and Wolfrum, J. and Brand, L. and Eggeling, C. and Seidel, C. A. M.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400050118}, pages = {517-523}, publisher = {Springer Berlin / Heidelberg}, title = {Detection and characterization of single molecules in aqueous solution}, volume = 63, year = 1996 }

  %0 Journal Article %1 zander1996detection %A Zander, C. %A Sauer, M. %A Drexhage, K. H. %A Ko, D.-S. %A Schulz, A. %A Wolfrum, J. %A Brand, L. %A Eggeling, C. %A Seidel, C. A. M. %D 1996 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 517-523 %T Detection and characterization of single molecules in aqueous solution %U http://dx.doi.org/10.1007/s003400050118 %V 63 %X Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100&#114ps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79&#450.33&#114ns (RB) and 3.79&#450.38&#114ns (R6G) in aqueous solution.

• New fluorescent dyes in the red region for biodiagnostics. Sauer, M.; Han, K. T.; Müller, R.; Nord, S.; Schulz, A.; Seeger, S.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Zander, C.; Drexhage, K. H. In Journal of Fluorescence, 5(3), S. 247–261. Springer Netherlands, 1995.
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  The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

  @article{springerlink:10.1007/BF00723896, abstract = {The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.}, author = {Sauer, M. and Han, K. T. and Müller, R. and Nord, S. and Schulz, A. and Seeger, S. and Wolfrum, J. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Zander, C. and Drexhage, K. H.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF00723896}, pages = {247-261}, publisher = {Springer Netherlands}, title = {New fluorescent dyes in the red region for biodiagnostics}, volume = 5, year = 1995 }

  %0 Journal Article %1 springerlink:10.1007/BF00723896 %A Sauer, M. %A Han, K. T. %A Müller, R. %A Nord, S. %A Schulz, A. %A Seeger, S. %A Wolfrum, J. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Zander, C. %A Drexhage, K. H. %D 1995 %I Springer Netherlands %J Journal of Fluorescence %P 247-261 %T New fluorescent dyes in the red region for biodiagnostics %U http://dx.doi.org/10.1007/BF00723896 %V 5 %X The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

• Sensitive fluorescence detection using laser diodes and multiplex dyes. Bachteler, G.; Drexhage, K.-H.; Arden-Jacob, J.; Han, K.-T.; Ko¨llner, M.; Mu¨ller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. In Journal of Luminescence, 60-61, S. 511–514. 1994.
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  A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.

  @article{Bachteler1994511, abstract = {A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.}, author = {Bachteler, G. and Drexhage, K.-H. and Arden-Jacob, J. and Han, K.-T. and Ko¨llner, M. and Mu¨ller, R. and Sauer, M. and Seeger, S. and Wolfrum, J.}, journal = {Journal of Luminescence}, keywords = {sauer}, note = {International Conference on Luminescence}, pages = {511 - 514}, title = {Sensitive fluorescence detection using laser diodes and multiplex dyes}, volume = {60-61}, year = 1994 }

  %0 Journal Article %1 Bachteler1994511 %A Bachteler, G. %A Drexhage, K.-H. %A Arden-Jacob, J. %A Han, K.-T. %A Ko¨llner, M. %A Mu¨ller, R. %A Sauer, M. %A Seeger, S. %A Wolfrum, J. %D 1994 %J Journal of Luminescence %P 511 - 514 %R DOI: 10.1016/0022-2313(94)90204-6 %T Sensitive fluorescence detection using laser diodes and multiplex dyes %U http://www.sciencedirect.com/science/article/B6TJH-46G3CGF-7R/2/13b4a8aed95422d6c11ea658258477ec %V 60-61 %X A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.
• Simultaneous antigen detection using multiplex dyes. Galla, K.; Arden-Jacob, J.; Deltau, G.; Drexhage, K. H.; Martin, M.; Sauer, M.; Wolfrum, J.; Seeger, S. In Journal of Fluorescence, 4(1), S. 111–115. Springer Netherlands, 1994.
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  To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.

  @article{galla1994simultaneous, abstract = {To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.}, author = {Galla, K. and Arden-Jacob, J. and Deltau, G. and Drexhage, K. H. and Martin, M. and Sauer, M. and Wolfrum, J. and Seeger, S.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF01876665}, pages = {111-115}, publisher = {Springer Netherlands}, title = {Simultaneous antigen detection using multiplex dyes}, volume = 4, year = 1994 }

  %0 Journal Article %1 galla1994simultaneous %A Galla, K. %A Arden-Jacob, J. %A Deltau, G. %A Drexhage, K. H. %A Martin, M. %A Sauer, M. %A Wolfrum, J. %A Seeger, S. %D 1994 %I Springer Netherlands %J Journal of Fluorescence %P 111-115 %T Simultaneous antigen detection using multiplex dyes %U http://dx.doi.org/10.1007/BF01876665 %V 4 %X To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.
• Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes. Bachteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Han, K. T.; Köllner, M.; Müller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. In Journal of Luminescence, 62(3-4), S. 101–108. 1994.
  • [ Abstract ]
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  A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.

  @article{Bachteler1994101, abstract = {A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.}, author = {Bachteler, G. and Drexhage, K. H. and Arden-Jacob, J. and Han, K. T. and Köllner, M. and Müller, R. and Sauer, M. and Seeger, S. and Wolfrum, J.}, journal = {Journal of Luminescence}, keywords = {sauer}, number = {3-4}, pages = {101 - 108}, title = {Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes}, volume = 62, year = 1994 }

  %0 Journal Article %1 Bachteler1994101 %A Bachteler, G. %A Drexhage, K. H. %A Arden-Jacob, J. %A Han, K. T. %A Köllner, M. %A Müller, R. %A Sauer, M. %A Seeger, S. %A Wolfrum, J. %D 1994 %J Journal of Luminescence %N 3-4 %P 101 - 108 %R DOI: 10.1016/0022-2313(94)90336-0 %T Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes %U http://www.sciencedirect.com/science/article/B6TJH-46FHCFY-2/2/86c15278719c7f6788703c0fe8cd6023 %V 62 %X A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.

• Design of Multiplex Dyes. Sauer, Markus; Schulz, Andreas; Seeger, Stefan; Wolfrum, Jürgen; Arden-Jacob, Jutta; Deltau, Gerhard; Drexhage, Karl H. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1734–1737. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
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  Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.

  @article{BBPC:BBPC19930971240, abstract = {Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.}, author = {Sauer, Markus and Schulz, Andreas and Seeger, Stefan and Wolfrum, Jürgen and Arden-Jacob, Jutta and Deltau, Gerhard and Drexhage, Karl H.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1734–1737}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Design of Multiplex Dyes}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971240 %A Sauer, Markus %A Schulz, Andreas %A Seeger, Stefan %A Wolfrum, Jürgen %A Arden-Jacob, Jutta %A Deltau, Gerhard %A Drexhage, Karl H. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1734--1737 %R 10.1002/bbpc.19930971240 %T Design of Multiplex Dyes %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971240/abstract %V 97 %X Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.
• New fluorescent labels for time-resolved detection of biomolecules. Sauer, M.; Han, K-T; Müller, R.; Schulz, A.; Tadday, R.; Seeger, S.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H. In Journal of Fluorescence, 3(3), S. 131–139. Springer Netherlands, 1993.
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  New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.

  @article{sauer1993fluorescent, abstract = {New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.}, author = {Sauer, M. and Han, K-T and Müller, R. and Schulz, A. and Tadday, R. and Seeger, S. and Wolfrum, J. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Drexhage, K. H.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF00862730}, pages = {131-139}, publisher = {Springer Netherlands}, title = {New fluorescent labels for time-resolved detection of biomolecules}, volume = 3, year = 1993 }

  %0 Journal Article %1 sauer1993fluorescent %A Sauer, M. %A Han, K-T %A Müller, R. %A Schulz, A. %A Tadday, R. %A Seeger, S. %A Wolfrum, J. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Drexhage, K. H. %D 1993 %I Springer Netherlands %J Journal of Fluorescence %P 131-139 %T New fluorescent labels for time-resolved detection of biomolecules %U http://dx.doi.org/10.1007/BF00862730 %V 3 %X New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.
• Biodiagnostics and Polymer Identification with Multiplex Dyes. Seeger, S.; Bachteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Deltau, G.; Galla, K.; Han, K. T.; Müller, R.; Köllner, M.; Rumphorst, A.; Sauer, M.; Schulz, A.; Wolfrum, J. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1542–1547. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
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  Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.

  @article{BBPC:BBPC19930971208, abstract = {Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.}, author = {Seeger, S. and Bachteler, G. and Drexhage, K. H. and Arden-Jacob, J. and Deltau, G. and Galla, K. and Han, K. T. and Müller, R. and Köllner, M. and Rumphorst, A. and Sauer, M. and Schulz, A. and Wolfrum, J.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1542–1547}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Biodiagnostics and Polymer Identification with Multiplex Dyes}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971208 %A Seeger, S. %A Bachteler, G. %A Drexhage, K. H. %A Arden-Jacob, J. %A Deltau, G. %A Galla, K. %A Han, K. T. %A Müller, R. %A Köllner, M. %A Rumphorst, A. %A Sauer, M. %A Schulz, A. %A Wolfrum, J. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1542--1547 %R 10.1002/bbpc.19930971208 %T Biodiagnostics and Polymer Identification with Multiplex Dyes %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971208/abstract %V 97 %X Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.
• Time-Resolved Fluorescence Studies of Labelled Nucleosides. Han, K.-T.; Sauer, M.; Schulz, A.; Seeger, S.; Wolfrum, J. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1728–1730. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
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  Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.

  @article{BBPC:BBPC19930971238, abstract = {Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.}, author = {Han, K.-T. and Sauer, M. and Schulz, A. and Seeger, S. and Wolfrum, J.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1728–1730}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Time-Resolved Fluorescence Studies of Labelled Nucleosides}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971238 %A Han, K.-T. %A Sauer, M. %A Schulz, A. %A Seeger, S. %A Wolfrum, J. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1728--1730 %R 10.1002/bbpc.19930971238 %T Time-Resolved Fluorescence Studies of Labelled Nucleosides %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971238/abstract %V 97 %X Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.