Fluorescence microscopy of living cells is a very powerful technique widely used in biomedical research. The revolution in the use of fluorescent proteins (the GFP family) and other fluorescent probes to tag specific molecules within living cells may yield information more specific than colocalization to a compartment. The direct interaction of two different species or the conformation of a single species that is multi-tagged may be measured by fluorescence resonance energy transfer (FRET). In the latter case, high speed widefield or confocal techniques may be employed to measure FRET interactions. However, intensity-based FRET microscopy is limited when used to study protein-protein interactions due to spectral bleed-through and unknown concentrations of the interacting proteins. Fluorescence lifetime imaging microscopy (FLIM) is independent of change in protein concentration and can yield information on the molecular microenvironment of a fluorescent tagged protein. Therefore FLIM in combination with FRET is a powerful tool to determine protein-protein interactions with high spatial and temporal resolution. The FLIM system proposed here will be part of a multi-user facility and cannot be used exclusively by one laboratory. The purpose of this application is to provide technology to the AECOM community which is not currently available. None of the equipment currently on campus is suited to the pulsed high speed low light imaging of live material for FLIM measurements. The common scientific need of the major users in this application is the ability to obtain high spatial and temporal resolution images from live cells for quantitative FLIM measurements. As discussed in the budget justification, the system we request will solve the problems of speed, sensitivity, and quantitation for an expert user group.
