Total Internal Reflection Fluorescence Microscope

Danuser, Gaudenz

Abstract

We request funds to purchase a Total Internal Reflection Fluorescence (TIRF) microscope with the capability for imaging 3 and 4 spectrally distinct fluorophores near-simultaneously (<50ms switching time between colors). No such instrument is currently available on the campus of the Harvard Medical School. We have five major users. Applications range from single molecule tracking of individual molecular motors to imaging of molecular dynamics of reconstituted cytoskeleton assemblies, cell surface receptors, and intracellular signal transduction using FRET and/or ratiometric biosensors. Seven minor users add further applications with diverse needs, justifying the acquisition of a flexible and modular state-of-the-art instrument. The microscope will be equipped with 7 laser lines. Using a TIRF-illuminator with fast motorized alignment of the fiber exit aperture relative to the objective back focal plane, true TIRF illumination can be achieved for all colors quasi-simultaneously. The color combinations for a particular experiment are in this case restricted only by the multi-bandpass characteristic of available dichroic mirrors. The motorized alignment of the fiber exit aperture can also be exploited to alter the depth of the TIRF field on the fly. We propose to equip the instrument with a conventional cooled CCD camera with small pixel size, appropriate for imaging macromolecular assemblies and biosensors, and with an electron-multiplying CCD camera, providing exquisite sensitivity for single molecule detection but coarser spatial resolution. Each of these proposed features and accessories has been thoroughly evaluated in the lab of the PI during a month long side-by-side comparison of TIRF microscopes with multi-spectral imaging capacity. The instrument will be installed and administered by highly-experienced staff of the shared microscope imaging facility of the Departments of Cell Biology, Systems Biology, and Biochemistry &Molecular Pharmacology. Appropriate space in the facility has been set aside. This arrangement will be optimal for the continued training of users and maintenance, warranting long-term use of this instrument investment.