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- W2015547834 abstract "The recent invention of super-resolution fluorescence microscopy allows nanoscopic investigation of cellular structures. Among these techniques, the Stochastic Optical Reconstruction Microscopy (STORM) is based on precise single molecule localization of photoswitchable fluorescent probes. By stochastically activating, imaging and deactivating subsets of fluorophores, it makes their images optically resolvable and determines their positions with nanometer precision. A super-resolution image is then reconstructed using these localizations. We now extend this approach to three-dimensional (3D) microscopy by determining the 3D coordinates of activated probes through astigmatism imaging: a cylindrical lens is inserted into the imaging optical path such that the image of individual molecules appear elliptical with the ellipticity depending on its z-position. Using this approach, we have achieved an optical resolution of 20-30 nm in the x-y direction and 50-60 nm in the z direction, representing an order of magnitude improvement over conventional fluorescence microscopy in all three dimensions. We have resolved the nanoscopic morphology of cellular structures that was previously deemed impossible by light microscopy.As a specific application, we use STORM to study the mechanism of clathrin mediated endocytosis in an in vitro reconstituted system. Proteins of interest in this system are directly labeled with photoswitchable fluorescent probes. This procedure is facilitated by covalently linking the two components of the probe, an activator dye and a photoswitchable reporter fluorophore, to form a single chemical unit prior to protein labeling. Using multicolor 3D STORM, we have characterized the spatial organization of plasma membrane, clathrin, actin and tubule forming proteins dynamin at the site of clathrin mediated endocytosis. These results reveal the molecular architecture of the nascent clathrin-coated pits at the nanometer scale and help to establish the role of actin and dynamin in membrane invagination, scission and vesicle formation." @default.
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- W2015547834 date "2009-02-01" @default.
- W2015547834 modified "2023-09-28" @default.
- W2015547834 title "Three-dimensional Super-resolution Fluorescence Microscopy and Its Application to Clathrin Mediated Endocytosis" @default.
- W2015547834 doi "https://doi.org/10.1016/j.bpj.2008.12.987" @default.
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