The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
W. E. Moerner - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.
Developmental Cell, 2019Co-Authors: Leonhard Mockl, Kayvon Pedram, Anish R. Roy, Venkatesh Krishnan, Anna-karin Gustavsson, Oliver Dorigo, Carolyn R. Bertozzi, W. E. MoernerAbstract:Summary The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and Super-Resolution localization Microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10–20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
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3d single molecule super resolution Microscopy with a tilted light sheet
Nature Communications, 2018Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D Super-Resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D Super-Resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
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3d single molecule super resolution Microscopy with a tilted light sheet
bioRxiv, 2017Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a tilted light sheet illumination scheme and engineered point spread functions with axial ranges up to ten microns for low-background, 3D super-localization of single molecules. This system is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells.
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single molecule spectroscopy imaging and photocontrol foundations for super resolution Microscopy nobel lecture
Angewandte Chemie, 2015Co-Authors: W. E. MoernerAbstract:The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later Super-Resolution Microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the Super-Resolution imaging of Eric Betzig and others, a new frontier for optical Microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.
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light paves the way to single molecule detection and photocontrol foundations of super resolution Microscopy
Imaging and Applied Optics 2015 (2015) paper JW1A.1, 2015Co-Authors: W. E. MoernerAbstract:More than 25 years ago, low temperature experiments aimed at establishing the ultimate limits to optical storage in solids led to the first optical detection and spectroscopy of a single molecule in the condensed phase. At this unexplored ultimate limit, many surprises occurred where single molecules showed both spontaneous changes (blinking) and light-driven control of emission, properties that were also observed in 1997 at room temperature with single green fluorescent protein variants. These observations form foundations for Super-Resolution Microscopy beyond the diffraction limit with single molecules, and tracking of single molecules in cells continues to yield surprises.
Anna-karin Gustavsson - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx.
Developmental Cell, 2019Co-Authors: Leonhard Mockl, Kayvon Pedram, Anish R. Roy, Venkatesh Krishnan, Anna-karin Gustavsson, Oliver Dorigo, Carolyn R. Bertozzi, W. E. MoernerAbstract:Summary The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and Super-Resolution localization Microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10–20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.
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3d single molecule super resolution Microscopy with a tilted light sheet
Nature Communications, 2018Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D Super-Resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D Super-Resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
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3d single molecule super resolution Microscopy with a tilted light sheet
bioRxiv, 2017Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a tilted light sheet illumination scheme and engineered point spread functions with axial ranges up to ten microns for low-background, 3D super-localization of single molecules. This system is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells.
Kai Johnsson - One of the best experts on this subject based on the ideXlab platform.
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small molecule fluorescent probes for live cell super resolution Microscopy
Journal of the American Chemical Society, 2019Co-Authors: Lu Wang, Michelle S Frei, Aleksandar Salim, Kai JohnssonAbstract:Super-Resolution fluorescence Microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to Super-Resolution Microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell Super-Resolution Microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell Super-Resolution Microscopy are given.
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small molecule fluorescent probes for live cell super resolution Microscopy
Journal of the American Chemical Society, 2019Co-Authors: Lu Wang, Michelle S Frei, Aleksandar Salim, Kai JohnssonAbstract:Super-Resolution fluorescence Microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to Super-Resolution Microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell Super-Resolution Microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell Super-Resolution Microscopy are given.
Michelle S Frei - One of the best experts on this subject based on the ideXlab platform.
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photoactivation of silicon rhodamines via a light induced protonation
Nature Communications, 2019Co-Authors: Michelle S Frei, Jan Ellenberg, Philipp Hoess, Marko Lampe, Bianca Nijmeijer, Moritz Kueblbeck, Hubert Wadepohl, Jonas RiesAbstract:Photoactivatable fluorophores are important for single-particle tracking and Super-Resolution Microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization Microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell Super-Resolution Microscopy. Activatable fluorophores are of interest for a wide range of applications but the need for caging groups complicates their development and application. Here, the authors report on a photoactivatable silicon rhodamine derivative and its application in live cell imaging and single-particle tracking.
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small molecule fluorescent probes for live cell super resolution Microscopy
Journal of the American Chemical Society, 2019Co-Authors: Lu Wang, Michelle S Frei, Aleksandar Salim, Kai JohnssonAbstract:Super-Resolution fluorescence Microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to Super-Resolution Microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell Super-Resolution Microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell Super-Resolution Microscopy are given.
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small molecule fluorescent probes for live cell super resolution Microscopy
Journal of the American Chemical Society, 2019Co-Authors: Lu Wang, Michelle S Frei, Aleksandar Salim, Kai JohnssonAbstract:Super-Resolution fluorescence Microscopy is a powerful tool to visualize biomolecules and cellular structures at the nanometer scale. Employing these techniques in living cells has opened up the possibility to study dynamic processes with unprecedented spatial and temporal resolution. Different physical approaches to Super-Resolution Microscopy have been introduced over the last years. A bottleneck to apply these approaches for live-cell imaging has become the availability of appropriate fluorescent probes that can be specifically attached to biomolecules. In this Perspective, we discuss the role of small-molecule fluorescent probes for live-cell Super-Resolution Microscopy and the challenges that need to be overcome for their generation. Recent trends in the development of labeling strategies are reviewed together with the required chemical and spectroscopic properties of the probes. Finally, selected examples of the use of small-molecule fluorescent probes in live-cell Super-Resolution Microscopy are given.
Yoav Shechtman - One of the best experts on this subject based on the ideXlab platform.
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3d single molecule super resolution Microscopy with a tilted light sheet
Nature Communications, 2018Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D Super-Resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D Super-Resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.
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3d single molecule super resolution Microscopy with a tilted light sheet
bioRxiv, 2017Co-Authors: Anna-karin Gustavsson, Petar N Petrov, Yoav Shechtman, W. E. MoernerAbstract:Tilted light sheet Microscopy with 3D point spread functions (TILT3D) combines a tilted light sheet illumination scheme and engineered point spread functions with axial ranges up to ten microns for low-background, 3D super-localization of single molecules. This system is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D Super-Resolution imaging with tens of nm localization precision throughout thick mammalian cells.
