Multi-Excitation TIRF Microscope for Shared Resource

Wells, K.

Abstract

We request funds for the purchase of a shared Multi-Excitation TIRF Microscope based on an inverted Nikon Eclipse Ti-E, configured to resolve details of dynamic macromolecular interactions near living cell surfaces. The instrument will be used to visualize and measure vesicular trafficking processes, protein- protein interactions, and plasma membrane transcytosis. These data are critical to multiple investigations related to, cardiac embryogenesis, cancer cell invasion, pancreatic ?-cell signaling and secretion, caveolae trafficking defects in endothelial cells, neuronal morphogenesis and remodeling, and molecular neurophysiology. Requisite components for live cell imaging experiments include, a 60X 1.49NA TIRF lens, stable TIRF illuminator with 4-channel multi-laser module aligned for multiple critical angles with dual-ports for simultaneous photo-activation and imaging, a high-speed precision autofocus system, very fast, high-resolution camera, and microscope incubation system. Eight Major User groups made up of investigators from four departments within the Vanderbilt University Medical Center (VUMC) use imaging techniques to study a broad range of problems in diabetes, cancer, heart disease, and neuronal development. All have considerable expertise with imaging-based approaches to quantify vesicular and non-vesicular processes and have critical experimental needs for multi-excitation TIRF imaging. This instrument has been rigorously configured based on the needs of the major user group with significant consideration given to optimal TIRF objective lens, critically aligned, rapidly modulated excitation sources, focus compensation, and high-speed image acquisition at the diffraction-limit, while maintaining live cell physiological conditions. The specifications are fundamental to the successful implementation of the multi-excitation TIRF imaging described in the Major User projects. The instrument will be located in Light Hall (LH 742) and maintained by the Cell Imaging Shared Resource (CISR). This room is a CISR microscopy facility, maintained exclusively by the CISR, for the benefit of all Vanderbilt investigators. This space is secure and easily accessible to all major users. The expertise and institutional support for this instrument are exceptional. Drs. Piston and Wells, Scientific Advisor and Director of the CISR, respectively, are world-renowned microscopy experts with confocal, multi-photon-excited fluorescence, FRAP, FRET, TIRF and other advanced imaging methods. The CISR has a 20-year history of assisting over 300 investigator labs with cutting-edge imaging expertise and cooperative research support. The VUMC has committed $50,000 to support the long-term operation of the instrument (see letter from Susan Wente, Associate Vice Chancellor for Research).