TIRFM-imaging system for in vitro and in vivo cell biology

Gladfelter, Amy

Abstract

The goal of this proposal is to acquire a total internal reflection (TIRF) microscope capable of simultaneous two-color acquisition (green, red) and near-simultaneous acquisition of a third color (far-red). The microscope would also be equipped with photobleaching/photoconversion capability. This equipment would be used as a primary research tool for quantitative analysis of dynamic processes, both biochemically and in live cells. There are five major users in the Department of Biological Sciences (Dartmouth College) and Department of Biochemistry (Geisel School of Medicine at Dartmouth) whose research projects are aimed at understanding molecular mechanisms of cellular processes such as actin dynamics, septin dynamics, intraflagellar transport during cilliary function, COPII transport from endoplasmic reticulum exit sites, and the function of a novel cytoskeletal element (Pil1 filaments). In all cases, multiple cellular elements are undergoing dynamics at sub-second timescales, including cytoskeleton and cellular membranes. Attainment and expansion of the goals of the funded research by these investigators requires the ability to monitor the dynamics of multiple molecules at high spatial and time resolution, and at single-molecule sensitivity. By limiting the excitation depth, TIRF microscopy has become a widely used technique to attain high spatial resolution and increased detection sensitivity. The iXON cameras and associated equipment allows simultaneous capture of two fluorescent signals, a necessity for processes with sub-second dynamics. In addition, a third fluorescent signal can be detected within 0.2 seconds of the other two signals. The EM-CCD camera allows single molecule sensitivity at moderate laser powers, minimizing photobleaching. This instrument would be the only one of its kind at Dartmouth and would serve the entire Dartmouth life science community.