Fluorescence Lifetime Imaging Microscopy (FLIM)

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DESCRIPTION (provided by applicant): We are requesting funds to purchase an Alba fluorescence lifetime imaging microscopy (FLIM) that will be housed in the University of Maryland-Baltimore Center for Fluorescence Spectroscopy (CFS), a campus-wide resource. The FLIM is a highly advanced spectroscopic method to image cells and tissues in which the image contrast is based on lifetime rather than fluorescence intensity. FLIM is now widely used for quantitative studies of cell function including tissue morphology and high density protein arrays. Resolution of intracellular structures is possible in images at high temporal and spatial resolution using fluorescently labeled components. We are requesting the Alba-FLIM instrument from ISS, Inc in Champaign, Illinois. We selected the Alba- FLIM instrument as a workhorse instrument suitable for multi-users environment that is robust and easy to use. Importantly, it is the only available instrument which can collect either time-domain (TD) or frequency-domain (FD) lifetime images. The wavelengths provided by laser diode sources are suitable for various experiments and we are able to use the other lasers available in the CFS using the access port of the instrument, including our Ti:Sapphire system for two-photon FLIM capabilities and pulsed dye lasers. The laser scanning capabilities will provide high spatial resolution. The frame rates can be faster using the phase angles measurement (FD mode) because the photon flux can be higher and we can spend less time at each pixel. Images can be acquired with a 4 microsec dwell time; a 512x512 pixel FLIM image is acquired in 1 sec. The time- correlated single photon counting (TCSPC) acquisition electronics allows resolution of complex intensity decays. An impressive group of users (14 major users with R01, P01 and R21; 5 minor users with R01, R21) from the School of Medicine University of Maryland at Baltimore will use the FLIM. The research topics of research projects (22 total) are diverse; including 11 projects related to the use of FRET in studies of intracellular protein-protein interactions, 5 projects related to intracellular imaging of ions, pH and protein co-localization, and 6 projects related to studies of surface plasmon-fluorophore interactions and their applications to biological and clinical systems. This instrument will have a major positive impact on our NIH-sponsored research and also quantitative results on cellular functions will have a significant impact on the NIH-funded research. We believe it is important for students and postdoctoral associates at UMB to gain exposure to FLIM as an important method for investigating biomolecular interactions. Indeed, providing access to state-of-the-art instrumentation is a key aspect of the training mission at UMB. The new ITC will enhance the training of students supported by a number of NIH training grants.