PHYC500/2007F:Supporting material/Lippincott

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The development of fluorescent proteins as molecular tags over the past decade has spurred a revolution by allowing complex biochemical processes to be correlated with the functioning of proteins in living cells. Among the new fluorescent proteins are those that can be photoactivated. These molecules are invisible until activated by a specific wavelength of light, at which point they become brightly fluorescent. In this talk, results will be presented from two new techniques employing photoactivatable fluorescent proteins: in cellula pulse-chase analysis and photoactivated localization microscopy (PALM). Results using in cellula pulse-chase analysis have permitted the visualization of autophagosome biogenesis in living cells, addressing the origin and mechanism of proliferation of these key organelles. Results using PALM- a technique involving serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules to determine the centers of fluorescent emission of individual molecules- have permitted fluorescently-tagged proteins within plasma membrane subdomains to be optically resolved at mean separations of a few nanometers.