This application requests funds for the purchase of a Fluorescence Lifetime Imaging Microscope (FLIMscope) as a Shared Instrument for the In Vivo Imaging Shared Resource (IVISR) at the University of California, San Diego (UCSD) Moores Cancer Center. There is no dedicated, shared FLIMscope at UCSD or local institutions and it is a clear unmet need. We have assembled a dozen outstanding users, including Dr. Roger Tsien (Nobel Laureate), that have all expressed the significant benefit a FLIMscope would bring to their biomedical research and strongly support this application. NIH-funded users are from multiple departments at UCSD and from the neighboring La Jolla Institute for Allergy and Immunology (LIAI). Whereas standard fluorescence microscopes only image fluorescence intensity, a FLIMscope also images the fluorescence lifetime of fluorophores enabling the discrimination of spectrally overlapping fluorophores, the use of fluorescence lifetime environment-sensitive probes, robust measurement of Forster Resonance Energy Transfer (FRET) with FLIM FRET, and Fluorescence Lifetime Correlation Spectroscopy (FLCS). The users'research projects will be dramatically enhanced by access to a FLIMscope applying these techniques to a broad variety of topics. For example, discriminating autofluorescence from fluorescent proteins and probes;measuring the microenvironment pH, superoxide level, metallomatrix proteinase activity, and cell membrane potential;and monitoring protein-protein interactions, nanoparticle accumulation or cleavage, FRET-quenching of fluorescent microbubbles, caspase-8 kinetics, and neutrophil adhesion in sickle cell disease. The FLIMscope we chose is the MicroTime 200 by Picoquant. The configuration has 4 pulsed laser diodes from ~400 nm to ~800nm allowing a broad range of fluorophores to be excited. Time correlated Single Photon Counting (TCSPC) technology with two Single Photon Avalanche Diode (SPAD) detectors allows simultaneous fluorescence lifetime measurement of two different wavelength fluorophores. These are integrated into a research grade inverted confocal microscope capable of three-dimensional scanning over both a small (80 micron) and large (10 cm) range. The host computer controls data acquisition and has fluorescence lifetime analysis software. The MicroTime 200 is developed by Picoquant, the world leaders in fluorescence time domain technology, and has been designed and manufactured specifically and solely for FLIM. Consequently, the MicroTime 200 is currently the best choice for a FLIMscope. The IVISR, supported by the In Vivo Cancer and Molecular Imaging Center (ICMIC) at UCSD, is a comprehensive imaging facility for users at UCSD and local institutions. It has an established infrastructure for resource administration, recharge mechanisms, training IVISR imaging staff, and supporting users. This application is also endorsed by the Institute of Engineering and Medicine, the Center for Multiscale Imaging of Living Systems, the UCSD Cancer Center Microscopy core, and the UCSD Moores Cancer Center.
