DEVELOPMENT OF INSTRUMENTATION FOR MULTIPHOTON MICROSCOPY

Cornell University Ithaca, Ithaca NY

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Abstract

Fluorescence correlation spectroscopy is ideally suited for detection and analysis of single fluorescent molecules, as exemplified by time traces of fluorescence bursts of individual rhodamine B molecules diffusing through the excitation volume. FCS using two-photon excitation provides the advantage of a mathematically well-defined three-dimensional geometry of the focal volume. During each event of a single molecule diffusing through the excitation volume, the burst of fluorescence can be analyzed in respect to fast kinetic processes with high statistical accuracy. We investigated single OD-TMR molecules in cell membranes by FCS and burst analysis with a multichannel scalar.