DIGITAL IMAGING FLUORESCENCE MICROSCOPY FACILITY

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This proposal request direct costs for the purchase of instrumentation necessary to construct a multiuser, real-time, combined transmitted light and epifluorescence microscopy digital imaging facility in the Renal Division at Children's Hospital. Th main instrumentation requested includes a Zeiss Axiovert 35 microscope equipped with fluorescence and DIC optics, a Photon Technology International dual wavelength epifluorescence illumination system, a Panasonic laser disc recorder and an Image-1/AT image processing and analysis system integrated with a Dell Model 325 80386 computer. A group of seven major users consisting of faculty members at the Children's and Brigham and Women's hospitals have been identified. Each major user has NIH grant support. The imaging facility will enable the major users to greatly extend their NIH-supported studies and initiate new lines of investigation not currently possible using existing instrumentation. For example, the imaging system will allow Drs. Harris, Strange and Zeidel to use fluorescent antibodies and fluid phase markers to characterize for the first time the dynamics and control of ADH-induced water channel insertion and retrieval in tight epithelial cells. Drs. Strange and Hebert will be able to address fundamental problems of cell volume regulation in renal epithelial cells by directly correlating changes in cell pH and Ca2+ with changes in cell volume. Dr. Neutra, who has extensively utilized ultrastructural methods to characterize apical endocytosis in intestinal epithelial cells, will be able for the first time to study the dynamics of this process and will be able to begin to elucidate cellular sorting mechanisms. The capability of the system for rapid sequential DIC and fluorescence and multiple fluorophore imaging will allow Dr. Lux to map ankyrin distribution in single living cells and to localize ankyrin with respect to other membrane an cytoskeletal components such as vimentin, tubulin, the Na+/K+ ATPase, etc. Finally, Dr. Simmons will be able to quantify the cellular and subcellular distribution of Na+/K+ ATPase subunits in native tissues and cell lines, detect transient transfection of mutant Na+/K+ ATPase subunits in cell cultures and characterize the role of the Na+ pump in membrane protein recycling and endocytic vesicle acidification.