Funds are requested for a mode-locked Argon Ion laser, a high speed (10 GHz) red-sensitive microchannel plate (MCP) photomultiplier tube (PMT), and near-IR (NIR) dye laser. Fluorescence Spectroscopy (CFS). The CFS is a multi-user facility which provides access to state-of-the-art time (TD) and frequency-domain (FD) fluorescence instrumentation and analysis programs for research in biochemistry and biophysics. During the start-up phase of the CFS its users and collaborators relied primarily on the FD instrumentation in Dr. Lakowicz's laboratory. Since the CFS instrumentation was not yet completed, the single existing NdYAG pump laser was adequate. This situation has now changed. The CFS now has three instruments for time-resolved fluorescence; a GHz frequency-domain instrument, a time-correlated single photon counting instrument (TCSPC, TD) and a newly developed apparatus for fluorescence lifetime imaging (FLIM). The FLIM method allows the generation of two-dimensional images in which the contrast reflects the local fluorescence decay time. It is not possible or practical to operate these instruments with a single pump laser. The CFS instrument room and its optical table is already prepared for an Argon Ion laser, which will be used with existing dye lasers and with the requested NIR dye laser. The Argon-pumped dye lasers will be used primarily for FD and FLIM measurements, where intensity and pointing stability are critical for successful experiments. The existing NdYAG laser will be used as the laser pump for TCSPC, which is less sensitive to drifts in laser power. The high speed MCP PMT and NIR dye laser (Styryl 9M, 790-900 nm) will be used to support users and collaborators in studies of time-dependent photon migration. The CFS scientific group consists of both core users and collaborators from the University of Maryland, collaborators from other universities, and external users of the CFS. The Argon Ion laser will be used for 30 NIH supported projects, 9 NSF supported projects, plus about 9 other projects with national peer-reviewed support. The projects involve widely ranging topics, including studies of Ca2+- binding proteins, membranes, hemoglobin, distance distributions, and ATPases. And finally, there are several new projects and/or collaborators on time-resolved photon migration, ion distribution around polyelectrolytes, and the use of FLIM to image calcium in cardiac cells.
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