The understanding of signal transduction processes governing lymphocyte differentiation and function require not only a delineation of the precise chain of biochemical events, but also determination of the spatial and temporal organization of the pathways. It is the purpose of the Imaging Core to provide support for the Principal Investigators in investigations in this regard. In general, the Core will provide advice, training and access to equipment essential for the acquisition and analysis of cell structure at the light microscopical level. The Core supports four broad categoriesof microscopic techniques: 1) identification of the subcellular distribution of molecules using conventional light and confocal microscopy; 2) three-dimensional reconstruction of immunofluorescent-labeled cells using assembly of confocal optical planes or deconvolution protocols; 3) visualization of fluorescent fusion proteins in living cells and 4) fluorescence resonance energy transfer (FRET) in fixed and live cells. The last two represent the most specialized and powerful technique available in the Core, as it allows the determination of co-localization of multiple signaling molecules in real time in living cells. FRET will be utilized for both the assay in vivo of selected kinase and small G protein activities as well as for the co-localization of critical signaling intermediates.
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