Core B: Nanoscopy

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Project Summary/Abstract: Core B Nanoscopy Super-resolution light microscopy or nanoscopy (i.e. microscopy with nanometer-scale resolution) is defined as imaging with a resolution below the diffraction limit of conventional light microscopy, which is ~200 nm in the equatorial (xy) and ~500 nm in the axial (z) dimension. Recently developed super-resolution methods that allow light microscopy to image subcellular structure and molecular localization on the 10s of nm scale have revolutionized neurobiology by allowing neuroscientists to study the inner workings of glia, axons, dendrites, and synapses in unprecedented detail that had previously only been possible using electron microscopy (EM). In particular, super-resolution fluorescence imaging has many advantages over EM including much easier sample preparation and staining procedures as well as the ability to be applied to not only fixed, but also living cells and tissues. Several different super-resolution fluorescence imaging methods have been developed, such as STimulated-Emission Depletion (STED) microscopy and the related methods of Stochastic Optical Reconstruction Microscopy (STORM) and Photo-activation Localization Microscopy (PALM). Each of these nanoscopy methods has advantages and limitations depending on the properties of the biological sample and fluorophores to be imaged. Thus, it is advantageous and even necessary to employ more than one of these different methods when investigating any given scientific question. Accordingly, the RMNDC Nanoscopy Core B will provide access to recently acquired instrumentation and technical support for NINDS-funded and other neuroscience investigators at the University of Colorado-Anschutz Medical Campus (UC-AMC) to perform state-of-the-art STED and STORM/PALM super-resolution imaging (Aim 1) and complementary Forster-resonance energy transfer (FRET)-based imaging (Aim 2).