The detection and imaging of molecules in biological systems is a vitally important tool in biomedicalresearch. Conventional fluorescence microscopy is an extremely useful technique, but is limited by the lossof resolution caused by emission from sources outside of the focal plane. Confocal Laser ScanningMicroscopy circumvents this problem by scanning the specimen with light from a laser-illuminated pinholeand imaging the emitted light through a detector pinhole. All out-of-focus fluorescence is rejected by thepinhole, resulting in submicron resolution in the X, Y, and Z axes.cIn 1998, funds from a NCRR Shared Instrument Grant, a UAB Health Services Foundation award, and otherresources from the UAB Office of the Provost, the Chairs of Departments in both the Medical and AcademicDivisions of DAB, and Center Directors were used to establish, the High Resolution Imaging Facility. Thisshared resource is equipped with a Leica Confocal Imaging Spectrophotometer TCS SP unit configured formultiphoton excitation with a Coherent MIRA Ti:Sa ultrafast laser with output selectable between 690-990nanometers 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 emissionspectrum to be delivered to the detector. The result is higher fluorescence yield, reduced crosstalk, andimproved image quality. The Facility also includes an Olympus Fluoview CLSM with fiber launched Kryptonion and Argon ion lasers, conventional fluorescence and DIC microscopy equipment, and work stationsequipped with digital deconvolution software, output and image storage devices. Through a cooperativeagreement with the Birmingham Veteran's Administration Medical Center, RDCC members have access to aLeica TCS SP unit equipped with a UV laser and UV corrected optics. The Leica instruments are equippedwith either three or four detector channels, a transmitted light channel, and Argon ion, Krypton ion andHeliunrNeon lasers, and are able to image up to three fluorophores simultaneously. In a cooperativeagreement with the Cell Adhesion and Matrix research center, and the Vision Science Research Center, theHRIF now offers access to a state-of-the-art FRET system. Significant funds for new in vivo imaginginstrumentation have been obtained through a successful NIH Blueprint application, and a SharedInstrument Grant application for high speed image acquisition capability is pending. The HRIF served 70RDCC investigators during the current funding period and their use accounted for 49% of the total facilityusage. During this same period, support from the RDCC P30 mechanism accounted for less than 20% of thetotal annual operating costs of the facility.
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