Imaging fluorescence from cells in 5-dimensions: x, y, wavelength, lifetime, and anisotropy.

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Fluorescent probes are widely used to study the location and function of biomolecules in cells. Using current fluorescence methods, the behavior of molecules within cells can be studied. Examples include: clustering, rotational motion, and intermolecular distance. These three examples are studied by measuring fluorescence lifetime and anisotropy. Simultaneous monitoring of the behavior of many types of molecules in cells over an extended area is an important unsolved physical problem. This proposal seeks to develop, study, and use a new approach to fluorescence imaging combining spectroscopy, fluorescence lifetime, and anisotropy with a goal of studying the behavior of many types of molecules in cells simultaneously. This will produce images with x and y spatial dimensions and fluorescence emission wavelength, fluorescence lifetime, and fluorescence anisotropy. The research has three parts: construction of new instrumentation, tests on defined mixtures of fluorescent compounds in a solution, and study of an important cell surface receptor. Successful completion of the study will provide biologists with new ways to study clustering, motion, and intermolecular distances of multiple molecular species in the field of a microscope.