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• 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.
  • [ Abstract ]
  • [ BibTeX ]
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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.

• 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.
  • [ Abstract ]
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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.
• 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.

• 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.
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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.
• 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.
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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.
• 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.
• 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 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.
• β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice. Tauscher, Sabine; Nakagawa, Hitoshi; Völker, Katharina; Werner, Franziska; Krebes, Lisa; Potapenko, Tamara; Doose, Sören; Birkenfeld, Andreas L.; Baba, Hideo A.; Kuhn, Michaela. In Cardiovascular Diabetology, 17, S. 103. 2018.
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  The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.

  @article{tauscher2018cellspecific, abstract = {The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.}, author = {Tauscher, Sabine and Nakagawa, Hitoshi and Völker, Katharina and Werner, Franziska and Krebes, Lisa and Potapenko, Tamara and Doose, Sören and Birkenfeld, Andreas L. and Baba, Hideo A. and Kuhn, Michaela}, journal = {Cardiovascular Diabetology}, keywords = {doose}, pages = 103, series = 1, title = {β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice}, volume = 17, year = 2018 }

  %0 Journal Article %1 tauscher2018cellspecific %A Tauscher, Sabine %A Nakagawa, Hitoshi %A Völker, Katharina %A Werner, Franziska %A Krebes, Lisa %A Potapenko, Tamara %A Doose, Sören %A Birkenfeld, Andreas L. %A Baba, Hideo A. %A Kuhn, Michaela %B 1 %D 2018 %J Cardiovascular Diabetology %P 103 %T β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice %U https://doi.org/10.1186/s12933-018-0747-3 %V 17 %X The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
• HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi. Brunk, Michael; Sputh, Sebastian; Doose, Sören; van de Linde, Sebastian; Terpitz, Ulrich. In Scientific Reports, 8(1), S. 605-. 2018.
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  The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.

  @article{brunk2018hyphatracker, abstract = {The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.}, author = {Brunk, Michael and Sputh, Sebastian and Doose, Sören and van de Linde, Sebastian and Terpitz, Ulrich}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {605–}, title = {HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi}, volume = 8, year = 2018 }

  %0 Journal Article %1 brunk2018hyphatracker %A Brunk, Michael %A Sputh, Sebastian %A Doose, Sören %A van de Linde, Sebastian %A Terpitz, Ulrich %D 2018 %J Scientific Reports %N 1 %P 605-- %R 10.1038/s41598-017-19103-1 %T HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi %U https://doi.org/10.1038/s41598-017-19103-1 %V 8 %X The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
• Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction. Haselmann, Holger; Mannara, Francesco; Werner, Christian; Planagumà, Jesús; Miguez-Cabello, Federico; Schmidl, Lars; Grünewald, Benedikt; Petit-Pedrol, Mar; Kirmse, Knut; Classen, Joseph; Demir, Fatih; Klöcker, Nikolaj; Soto, David; Doose, Sören; Dalmau, Josep; Hallermann, Stefan; Geis, Christian. In Neuron, 100(1), S. 91–105.e9. 2018.
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  AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.

  @article{haselmann2018human, abstract = {AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.}, author = {Haselmann, Holger and Mannara, Francesco and Werner, Christian and Planagumà, Jesús and Miguez-Cabello, Federico and Schmidl, Lars and Grünewald, Benedikt and Petit-Pedrol, Mar and Kirmse, Knut and Classen, Joseph and Demir, Fatih and Klöcker, Nikolaj and Soto, David and Doose, Sören and Dalmau, Josep and Hallermann, Stefan and Geis, Christian}, journal = {Neuron}, keywords = {werner}, number = 1, pages = {91–105.e9}, title = {Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction}, volume = 100, year = 2018 }

  %0 Journal Article %1 haselmann2018human %A Haselmann, Holger %A Mannara, Francesco %A Werner, Christian %A Planagumà, Jesús %A Miguez-Cabello, Federico %A Schmidl, Lars %A Grünewald, Benedikt %A Petit-Pedrol, Mar %A Kirmse, Knut %A Classen, Joseph %A Demir, Fatih %A Klöcker, Nikolaj %A Soto, David %A Doose, Sören %A Dalmau, Josep %A Hallermann, Stefan %A Geis, Christian %D 2018 %J Neuron %N 1 %P 91--105.e9 %R https://doi.org/10.1016/j.neuron.2018.07.048 %T Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction %U http://www.sciencedirect.com/science/article/pii/S0896627318306469 %V 100 %X AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.

• 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.
• 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.

• 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.
• 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.

• 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.

• 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.

• 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.
• 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.
• Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale. Steger, Katrin; Bollmann, Stefan; Noe, Frank; Doose, Soren. In Phys. Chem. Chem. Phys., S. -. The Royal Society of Chemistry, 2013.
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  Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.

  @article{C3CP50644D, abstract = {Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.}, author = {Steger, Katrin and Bollmann, Stefan and Noe, Frank and Doose, Soren}, journal = {Phys. Chem. Chem. Phys.}, keywords = {doose}, pages = {-}, publisher = {The Royal Society of Chemistry}, title = {Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale}, year = 2013 }

  %0 Journal Article %1 C3CP50644D %A Steger, Katrin %A Bollmann, Stefan %A Noe, Frank %A Doose, Soren %D 2013 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %P - %R 10.1039/C3CP50644D %T Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale %U http://dx.doi.org/10.1039/C3CP50644D %X Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.

• 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.
• Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding. Göhler, Antonia; Büchner, Claudia; André, Sabine; Doose, Sören; Kaltner, Herbert; Gabius, Hans-Joachim. In Biochimie, 94(12), S. 2649–2655. 2012.
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  Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.

  @article{Göhler20122649, abstract = {Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.}, author = {Göhler, Antonia and Büchner, Claudia and André, Sabine and Doose, Sören and Kaltner, Herbert and Gabius, Hans-Joachim}, journal = {Biochimie}, keywords = {doose}, number = 12, pages = {2649 - 2655}, title = {Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding}, volume = 94, year = 2012 }

  %0 Journal Article %1 Göhler20122649 %A Göhler, Antonia %A Büchner, Claudia %A André, Sabine %A Doose, Sören %A Kaltner, Herbert %A Gabius, Hans-Joachim %D 2012 %J Biochimie %N 12 %P 2649 - 2655 %R 10.1016/j.biochi.2012.08.001 %T Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding %U http://www.sciencedirect.com/science/article/pii/S0300908412003100 %V 94 %X Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.

• Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy. Gohler, Antonia; Buchner, Claudia; Andre, Sabine; Doose, Soren; Kaltner, Herbert; Gabius, H.-J. In Analyst, 136(24), S. 5270–5276. The Royal Society of Chemistry, 2011.
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  Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.

  @article{C1AN15692F, abstract = {Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.}, author = {Gohler, Antonia and Buchner, Claudia and Andre, Sabine and Doose, Soren and Kaltner, Herbert and Gabius, H.-J.}, journal = {Analyst}, keywords = {doose}, number = 24, pages = {5270-5276}, publisher = {The Royal Society of Chemistry}, title = {Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy}, volume = 136, year = 2011 }

  %0 Journal Article %1 C1AN15692F %A Gohler, Antonia %A Buchner, Claudia %A Andre, Sabine %A Doose, Soren %A Kaltner, Herbert %A Gabius, H.-J. %D 2011 %I The Royal Society of Chemistry %J Analyst %N 24 %P 5270-5276 %R 10.1039/C1AN15692F %T Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy %U http://dx.doi.org/10.1039/C1AN15692F %V 136 %X Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.
• 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.
• 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.
• 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.
• 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

• 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.
• 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.

• 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.

• Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides. Doose, Sören. In CHEMPHYSCHEM, 9(18), S. 2687–2689. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  @article{Doose2008, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören}, journal = {CHEMPHYSCHEM}, keywords = {doose}, month = 12, number = 18, pages = {2687-2689}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides}, type = {Editorial Material}, volume = 9, year = 2008 }

  %0 Journal Article %1 Doose2008 %A Doose, Sören %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 18 %P 2687-2689 %T Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides %U http://dx.doi.org/10.1002/cphc.200800545 %V 9
• 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.
• Reconfigurable, braced, three-dimensional DNA nanostructures. Goodman, Russell P.; Heilemann, Mike; Doose, Sören; Erben, Christoph M.; Kapanidis, Achillefs N.; Turberfield, Andrew J. In NATURE NANOTECHNOLOGY, 3(2), S. 93–96. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2008.
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  DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.

  @article{Goodman2008, abstract = {DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Goodman, Russell P. and Heilemann, Mike and Doose, Sören and Erben, Christoph M. and Kapanidis, Achillefs N. and Turberfield, Andrew J.}, journal = {NATURE NANOTECHNOLOGY}, keywords = {mike}, month = {02}, number = 2, pages = {93-96}, publisher = {NATURE PUBLISHING GROUP}, title = {Reconfigurable, braced, three-dimensional DNA nanostructures}, type = {Article}, volume = 3, year = 2008 }

  %0 Journal Article %1 Goodman2008 %A Goodman, Russell P. %A Heilemann, Mike %A Doose, Sören %A Erben, Christoph M. %A Kapanidis, Achillefs N. %A Turberfield, Andrew J. %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2008 %I NATURE PUBLISHING GROUP %J NATURE NANOTECHNOLOGY %N 2 %P 93-96 %T Reconfigurable, braced, three-dimensional DNA nanostructures %U http://dx.doi.org/10.1038/nnano.2008.3 %V 3 %X DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.

• 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.
• Periodic acceptor excitation spectroscopy of single molecules. Doose, Sören; Heilemann, Mike; Michalet, Xavier; Weiss, Shimon; Kapanidis, Achillefs. In European Biophysics Journal, 36(6), S. 669–674. Springer Berlin / Heidelberg, 2007.
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  Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

  @article{Doose2007d, abstract = {Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.}, author = {Doose, Sören and Heilemann, Mike and Michalet, Xavier and Weiss, Shimon and Kapanidis, Achillefs}, journal = {European Biophysics Journal}, keywords = {mike}, note = {10.1007/s00249-007-0133-7}, pages = {669-674}, publisher = {Springer Berlin / Heidelberg}, title = {Periodic acceptor excitation spectroscopy of single molecules}, volume = 36, year = 2007 }

  %0 Journal Article %1 Doose2007d %A Doose, Sören %A Heilemann, Mike %A Michalet, Xavier %A Weiss, Shimon %A Kapanidis, Achillefs %D 2007 %I Springer Berlin / Heidelberg %J European Biophysics Journal %P 669-674 %T Periodic acceptor excitation spectroscopy of single molecules %U http://dx.doi.org/10.1007/s00249-007-0133-7 %V 36 %X Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.
• 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.
  • [ Abstract ]
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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.
• 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.
  • [ Abstract ]
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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.
• 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.
• Optical amplification from single excitons in collodial quantum dots. Doose, Sören. In SMALL, 3(11), S. 1856–1858. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2007.
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  @article{Doose2007, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören}, journal = {SMALL}, keywords = {doose}, month = 11, number = 11, pages = {1856-1858}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Optical amplification from single excitons in collodial quantum dots}, type = {Editorial Material}, volume = 3, year = 2007 }

  %0 Journal Article %1 Doose2007 %A Doose, Sören %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2007 %I WILEY-V C H VERLAG GMBH %J SMALL %N 11 %P 1856-1858 %T Optical amplification from single excitons in collodial quantum dots %U http://dx.doi.org/10.1002/smll.200700478 %V 3

• Advances in fluorescence imaging with quantum dot bio-probes. Pinaud, F; Michalet, X; Bentolila, LA; Tsay, JM; Doose, S; Li, JJ; Iyer, G; Weiss, S. In BIOMATERIALS, 27(9), S. 1679–1687. ELSEVIER SCI LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND, 2006.
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  After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.

  @article{Pinaud2006, abstract = {After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, author = {Pinaud, F and Michalet, X and Bentolila, LA and Tsay, JM and Doose, S and Li, JJ and Iyer, G and Weiss, S}, journal = {BIOMATERIALS}, keywords = {doose}, month = {03}, number = 9, pages = {1679-1687}, publisher = {ELSEVIER SCI LTD}, title = {Advances in fluorescence imaging with quantum dot bio-probes}, type = {Article}, volume = 27, year = 2006 }

  %0 Journal Article %1 Pinaud2006 %A Pinaud, F %A Michalet, X %A Bentolila, LA %A Tsay, JM %A Doose, S %A Li, JJ %A Iyer, G %A Weiss, S %C THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND %D 2006 %I ELSEVIER SCI LTD %J BIOMATERIALS %N 9 %P 1679-1687 %T Advances in fluorescence imaging with quantum dot bio-probes %U http://dx.doi.org/10.1016/j.biomaterials.2005.11.018 %V 27 %X After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.
• Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy. Tsay, JM; Doose, S; Weiss, S. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(5), S. 1639–1647. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.

  @article{Tsay2006, abstract = {CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tsay, JM and Doose, S and Weiss, S}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {doose}, month = {02}, number = 5, pages = {1639-1647}, publisher = {AMER CHEMICAL SOC}, title = {Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 128, year = 2006 }

  %0 Journal Article %1 Tsay2006 %A Tsay, JM %A Doose, S %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 5 %P 1639-1647 %T Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ja056162i %V 128 %X CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.
• 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.

• Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis. Kapanidis, AN; Margeat, E; Laurence, TA; Doose, S; Ho, SO; Mukhopadhyay, J; Kortkhonjia, E; Mekler, V; Ebright, RH; Weiss, S. In MOLECULAR CELL, 20(3), S. 347–356. CELL PRESS, 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA, 2005.
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  We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70%-90%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50%-60%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.

  @article{Kapanidis2005, abstract = {We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70\%-90\%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50\%-60\%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.}, address = {1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA}, author = {Kapanidis, AN and Margeat, E and Laurence, TA and Doose, S and Ho, SO and Mukhopadhyay, J and Kortkhonjia, E and Mekler, V and Ebright, RH and Weiss, S}, journal = {MOLECULAR CELL}, keywords = {doose}, month = 11, number = 3, pages = {347-356}, publisher = {CELL PRESS}, title = {Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis}, type = {Article}, volume = 20, year = 2005 }

  %0 Journal Article %1 Kapanidis2005 %A Kapanidis, AN %A Margeat, E %A Laurence, TA %A Doose, S %A Ho, SO %A Mukhopadhyay, J %A Kortkhonjia, E %A Mekler, V %A Ebright, RH %A Weiss, S %C 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA %D 2005 %I CELL PRESS %J MOLECULAR CELL %N 3 %P 347-356 %T Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis %U http://dx.doi.org/10.1016/j.molcel.2005.10.012 %V 20 %X We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70%-90%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50%-60%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.
• 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.
• 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.
• Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy. Doose, S; Tsay, JM; Pinaud, F; Weiss, S. In ANALYTICAL CHEMISTRY, 77(7), S. 2235–2242. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.

  @article{Doose2005, abstract = {A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Doose, S and Tsay, JM and Pinaud, F and Weiss, S}, journal = {ANALYTICAL CHEMISTRY}, keywords = {doose}, month = {04}, number = 7, pages = {2235-2242}, publisher = {AMER CHEMICAL SOC}, title = {Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Doose2005 %A Doose, S %A Tsay, JM %A Pinaud, F %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 7 %P 2235-2242 %T Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ac050035n %V 77 %X A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.
• Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation. Tsay, JM; Doose, S; Pinaud, F; Weiss, S. In JOURNAL OF PHYSICAL CHEMISTRY B, 109(5), S. 1669–1674. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.

  @article{Tsay2005, abstract = {The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tsay, JM and Doose, S and Pinaud, F and Weiss, S}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {doose}, month = {02}, number = 5, pages = {1669-1674}, publisher = {AMER CHEMICAL SOC}, title = {Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation}, type = {Article}, volume = 109, year = 2005 }

  %0 Journal Article %1 Tsay2005 %A Tsay, JM %A Doose, S %A Pinaud, F %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 5 %P 1669-1674 %T Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation %U http://dx.doi.org/10.1021/jp046828f %V 109 %X The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.
• Quantum dots for live cells, in vivo imaging, and diagnostics. Michalet, X; Pinaud, FF; Bentolila, LA; Tsay, JM; Doose, S; Li, JJ; Sundaresan, G; Wu, AM; Gambhir, SS; Weiss, S. In SCIENCE, 307(5709), S. 538–544. AMER ASSOC ADVANCEMENT SCIENCE, 1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA, 2005.
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  Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

  @article{Michalet2005, abstract = {Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.}, address = {1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA}, author = {Michalet, X and Pinaud, FF and Bentolila, LA and Tsay, JM and Doose, S and Li, JJ and Sundaresan, G and Wu, AM and Gambhir, SS and Weiss, S}, journal = {SCIENCE}, keywords = {doose}, month = {01}, number = 5709, pages = {538-544}, publisher = {AMER ASSOC ADVANCEMENT SCIENCE}, title = {Quantum dots for live cells, in vivo imaging, and diagnostics}, type = {Review}, volume = 307, year = 2005 }

  %0 Journal Article %1 Michalet2005 %A Michalet, X %A Pinaud, FF %A Bentolila, LA %A Tsay, JM %A Doose, S %A Li, JJ %A Sundaresan, G %A Wu, AM %A Gambhir, SS %A Weiss, S %C 1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA %D 2005 %I AMER ASSOC ADVANCEMENT SCIENCE %J SCIENCE %N 5709 %P 538-544 %T Quantum dots for live cells, in vivo imaging, and diagnostics %U http://dx.doi.org/10.1126/science.1104274 %V 307 %X Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

• Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules. Kapanidis, AN; Lee, NK; Laurence, TA; Doose, S; Margeat, E; Weiss, S. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101(24), S. 8936–8941. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2004.
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  We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.

  @article{Kapanidis2004, abstract = {We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Kapanidis, AN and Lee, NK and Laurence, TA and Doose, S and Margeat, E and Weiss, S}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {doose}, month = {06}, number = 24, pages = {8936-8941}, publisher = {NATL ACAD SCIENCES}, title = {Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules}, type = {Article}, volume = 101, year = 2004 }

  %0 Journal Article %1 Kapanidis2004 %A Kapanidis, AN %A Lee, NK %A Laurence, TA %A Doose, S %A Margeat, E %A Weiss, S %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2004 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 24 %P 8936-8941 %T Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules %U http://dx.doi.org/10.1073/pnas.0401690101 %V 101 %X We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.

• The power and prospects of fluorescence microscopies and spectroscopies. Michalet, X; Kapanidis, AN; Laurence, T; Pinaud, F; Doose, S; Pflughoefft, M; Weiss, S. In ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, 32, S. 161–182. ANNUAL REVIEWS, 4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA, 2003.
  • [ Abstract ]
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  Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

  @article{Michalet2003, abstract = {Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.}, address = {4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA}, author = {Michalet, X and Kapanidis, AN and Laurence, T and Pinaud, F and Doose, S and Pflughoefft, M and Weiss, S}, journal = {ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE}, keywords = {doose}, pages = {161-182}, publisher = {ANNUAL REVIEWS}, title = {The power and prospects of fluorescence microscopies and spectroscopies}, type = {Review}, volume = 32, year = 2003 }

  %0 Journal Article %1 Michalet2003 %A Michalet, X %A Kapanidis, AN %A Laurence, T %A Pinaud, F %A Doose, S %A Pflughoefft, M %A Weiss, S %C 4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA %D 2003 %I ANNUAL REVIEWS %J ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE %P 161-182 %T The power and prospects of fluorescence microscopies and spectroscopies %U http://dx.doi.org/10.1146/annurev.biophys.32.110601.142525 %V 32 %X Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.