The objective of this application is to establish a novel state-of-the-art facility termed the High-speed Total Internal Reflection Fluorescence (TIRF) Microscope for the observation of fluorescent molecules within living cells. The envisioned system is centered on a Zeiss Axiovert 200M inverted microscope equipped with a laser-illuminated system for TIRF microscopy. The TIRF illumination is introduced to the system via a slider that inserts into the standard illumination beam path and allows simultaneous TIRF, epifluorescence and other modalities of microscopy. This system is equipped with two intensified, high-speed Princeton cameras, synchronized via a Stanford DG535 pulse generator, in order to provide a large field of view at high magnifications in multi-color experiments. Image acquisition is orchestrated by MetaMorph software (DIG). This system will be housed, maintained and made available with expert help in the Oklahoma Medical Research Foundation Core Facility for Imaging. The capabilities of this system are required to meet the demanding experimental designs of the members of the major user group, each of whom require TIRF to determine the behaviors of given molecules at the cell surface-substrate interface but in addition need to correlate these behaviors with additional markers at the cell surface or immediately internal to the cell. The members of the major user group will apply these imaging techniques to several areas: Membrane Tether Formation and P- and E-selectin Function in Leukocyte Rolling (Schmidtke, McEver), Microtubule Function at the Cleavage Furrow (Gorbsky), Cytoskeleton Function in GLUT4 Vesicle Trafficking, T Cell Membrane Rafts (Rodgers) and Intracellular Dynamics in Dictyostelium (Clarke). The High-speed TIRF Microscope will become a pivotal resource for the local community of researchers requiring TIRF, high-speed, high- resolution, ultrasensitive fluorescence microscopy in living cells. ? ? ?
