HIGH RESOLUTION MICROSCOPY

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DESCRIPTION (provided by applicant): The detection and imaging of molecules in biological systems is a vitally important tool in biomedical research. Conventional fluorescence microscopy is an extremely useful technique, but is limited by the loss of resolution caused by emission from sources outside of the focal plane. Confocal Laser Scanning Microscopy circumvents this problem by scanning the specimen with light from a laser-illuminated pinhole and imaging the emitted light through a detector pinhole. All out-of-focus fluorescence is rejected by the pinhole, resulting in submicron resolution in the X, Y, and Z axes. In 1998, Dr. Kent Keyser was the recipient of an NIH NCRR Shared Instrument Grant, a UAB Health Services Foundation award, and start-up funds from the UAB Office of the Provost, the Chairs of Departments in both the Medical and Academic Divisions of UAB, and Center Directors. These funds were used to establish a new facility, the High Resolution Imaging Facility. This imaging resource is equipped with a Leica Confocal Imaging Spectrophotometer TCS SP unit. It is configured for multiphoton excitation with a Coherent MIRA Ti:Sa ultrafast laser with output selectable between 690-990 nanometers. This permits imaging of living cells and tissues with minimal phototoxicity, and of structures 200-400 micrometers from the tissue surface. The SP allows user selection of specific portions of the emission spectrum to be delivered to the detector. The result is higher fluorescence yield, reduced crosstalk, and improved image quality. The Facility also includes an Olympus Fluoview CLSM with fiber launched Krypton ion and Argon ion lasers, conventional fluorescence and DIG microscopy equipment, and work stations equipped with digital deconvolution software, output and image storage devices. Through a cooperative agreement with the Birmingham Veteran's Administration Medical Center, Cancer Center members have access to a Leica TCS SP unit equipped with a UV laser and UV corrected optics. The Leica instruments are equipped with either three or four detector channels, a transmitted light channel, and Argon ion, Krypton ion and Helium:Neon lasers, and are able to image up to three fluorophores simultaneously. In a cooperative agreement with the Cell Adhesion and Matrix research center, and the Vision Science Research Center, the HRIF now offers access to a state of the art FRET system. The Facility offers Comprehensive Cancer Center members access to a comprehensive imaging facility with a full range of instrumentation and the expertise to use these resources productively.