CONFOCAL MICROSCOPY FACILITY FOR LIVING SPECIMENS

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The objective is to secure state-of-the-art confocal microscopy equipment to study living biological specimens ranging from in vivo preparations, to in vitro multicellular tissues, to the single cell and subcellular levels. Fifteen major NIH funded grants/projects will benefit from the nine major projects described that require this instrumentation. The aims of the nine projects are to assess: vascular Ca in simulated weightlessness (E.M. Hasser); synaptic vesicle exocytosis (M. Hay); in vivo and in vitro microvessel permeability (V.H. Huxley); Ca and K channel distribution (A.W. Jones); endothelial and smooth muscle Ca in microvessels (M.H. Laughlin); vascular cell Ca in chronic coronary occlusion (J.L Parker); Ca sparks in endotoxemic myocytes (L.J. Rubin); smooth muscle Ca in diabetes (M. Sturek); vascular growth factor receptors (R.L. Terjung). Our needs are to: 1) adapt microscope systems to use living in vivo, in vitro tissue and single coli preparations, 2) combine confocal studies with electrophysiology and microinjection techniques, 3) have sufficient time resolution to resolve dynamic events. The existing facility is inadequate for our needs because: 1) it mainly accomodates chemically fixed preparations, 2) it serves typically 5 major users per week, thus creating a severe shortage of large time blocks required for our NIH funded microscopy-based research on living specimens, and 3) low time resolution (2 s/image). The overall organizational plan is to purchase a Noran Odyssey (acquisition 33 ms/image) for in vivo and in vitro multicellular tissue studies, which is enabled by adaptation of the stage, etc. Dual emission wide-field epifluorescence capabilities will be included for versatility. A Noran OZ will enable primarily single cell imaging of Ca dynamics at 4 ms/image, 500 times faster than in the existing facility. The investigators to use the proposed facility have extensive experience in microscopic study of living specimens. Two data analysis stations on a network will provide further quantitation of colocalization and proximity of molecules, comparison of confocal images to deconvolution methods, and 3-dimensional reconstruction. Technical support will be provided by the University for two years for maintenance of these ongoing research programs. It is anticipated that these major users will represent greater than 85 percent of the total usage of the requested equipment. The remaining time will be provided to other research groups in the biomedical area on campus. This confocal equipment will enable full implementation of the novel and versatile adaptations proposed and will substantially enhance these research projects. Our major strength is integration of techniques to study living specimens at single cell and multicellular levels.