Funds are requested for the purchase of a confocal laser scanning microscope (CLSM) with accompanying computer hardware and software necessary for data storage and for the 3-dimensional reconstruction of biological material. Currently, fluorescent light microscopes are limited in their resolution of thick biological specimens (eg. whole cells or tissues) by the fluorescence contributions from off- focus layers in the specimen contributing to the image formation. ln short, the relatively long depth of focus precludes submicron resolution within specimens exceeding 5.10 um thickness. An approach to this problem used by the CSLM is to both optimally illuminate with a point source of light and to image with a point detector the identical spot in the specimen. By then scanning the specimen in X, Y and Z axes and combining computerized data storage with image reconstruction programs it is feasible to generate dimensional images of the fluorescent specimen with improvements in resolution of about 1.3 times that predicted from conventional light microscopes. Specifically we request monies to purchase the Biorad-Polaron Laser Sharp Confocal System with an argon laser, a photomultiplier, an AT compatible PC with scanning control board and image acquisition board. In addition, funds will be needed to add a Z-axis focusing system, a TV monitor and camera system, an image reconstruction program developed at the MRC Laboratory for Molecular Biology (Cambridge, England), dual channel additions for 2-wavelength analyses (FITC and Rhodamine), optical disc storage facilities, scanning transmission assembly for recording of parallel transmitted light images and an anti-vibration table. Finally, we request the purchase of a Zeiss Axioplan fluorescence microscope. The facility will be used as a core laboratory shared by Drs. Fischman, Rodriguez-Boulan, Wall (Department of Cell Biology and Anatomy), and Townes-Anderson (Department of Physiology). All investigators are independently funded by NSF or NIH research grants but lack the equipment funds to purchase this state-of-the- art equipment.
