PHONE CONSULTATION: CONFOCAL MICROSCOPY

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Talk at DAMOP meeting, Santa Fe May 27-30 1998 Session I1 - Optical Techniques in Molecular Biology. NVITED session, Friday morning, May 29 FCS, a technique introduced in 1972 (Magde et al. 1972, Appl. Phys. Lett. 29:705), is currently being employed for ultrasensitive studies of molecular diffusion and aggregation, photophysical dynamics and fast chemical reactions. When performed in confocal geometries with one- or two-photon excitation (Denk et al. 1990, Science 248:73) of photostable dyes, its sensitivity enables us to follow the dynamics of sparse biologically relevant agents such as drugs and pathogens in solution as well as in living cells. Minimally interfering with biological functions of the observed system, FCS allows us to address a variety of important questions in molecular biology. We report that green fluorescent protein (GFP), a popular tool for in-vivo studies, is not only well suited for intracellular FCS, but moreover turned out to be a perfect candidate for the study of intramolecular dynamics. It exhibits a rapid flickering in the 100 ?s time range that we have identified to be due to reversible proto nation of the chromophore. Supported by NIH-P41.RR04224, NSF-D1R 8800278