ANALYSIS OF NEAR-MEMBRANE CALCIUM USING TIRFM AND NSOM

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Many in vitro experiments demonstrate that the transient calcium concentrations needed to trigger cellular events such as signaling cascades and synaptic transmission are actually much higher than the average resting concentrations of calcium experimentally measured in the cytoplasm of the cell. These findings suggest that calcium reaches transient concentrations that are much higher than that measured in the bulk of the cell. We propose to utilize two optical imaging methods to obtain better resolution of near- membrane dynamics and hotspots: Total Internal Reflection Fluorescence Microscopy (TIRFM) and Near-field Scanning Optical Microscopy (NSOM). Both of these techniques effectively excite only 100 -200 nm into the bulk of the cell. TIRFM will provide a high resolution method of imaging near the membrane of a neuroblastoma cell, and NSOM will monitor a single channel with fast temporal resolution. The combination of the two techniques will elucidate the dynamics of calcium microdomains, including information on their size, concentration, location, and lifetime.