UNM SUPER-RESOLUTION CORE

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Over the last two years, fluorescence microscopy has experienced an order of magnitude improvement in resolution via single molecule based imaging techniques [296]. Evolution in the technologies of detectors, computational power, molecular biology and fluorescence probes now allows the brute force approach of localizing thousands of individual probes that are labeling specific targets in vivo. The basic concept of this imaging technique is that single molecules can be localized with an accuracy that scales roughly as sigma(loc)=sigma psf/N[1/2], where sigma (ioc) is the localization accuracy sigma psf is the width of the microscope point spread function, and N is the expected number of collected photons from the single molecule [297]. Recent developments in bright photo-activatable, photo-switchable and naturally intermittent fluorescent probes have allowed high densities of single fluorescent molecules to be imaged and localized individually with accuracy approaching 10 nm, leading to super-resolution images, in fixed or neariy static structures. This concept is illustrated in Figure 56. SML-SR has been demonstrated by several groups [26,298,299] with variations based on the probes used, imaging conditions and analysis approaches. Single Molecule Localization based Super-Resolution (SML-SR) techniques join SPT and single molecule imaging as the most powerful available tools to access protein-protein dynamics at the 10 nm scale in live and fixed cells. One important advantage of SML-SR over other SR approaches (such as STED [300], and Saturated Patterned Excitation [301] is that positions of labeled targets (used to generate the SR images) can be used directly for analysis of clustering and co-clustering as done with immuno-gold labeling in EM [11]. The primary goals of the SR-core are to (1) provide these state of the art single molecule fluorescence techniques to the STMC and (2) develop new techniques that allow access to ever smaller spatial and temporal scales. The SR-core personnel will work closely with the biologists and the analysis core to design and execute single molecule experiments to directly answer questions described elsewhere in this proposal.