We propose to establish a confocal fluorescence microscopy facility at the Lawrence E. Martin Laboratories of the Massachusetts General Hospital (MGH), by purchase of a BioRad MRC Lasersharp Confocal Imaging System and a Zeiss Axiophot microscope, The facility will be used for both qualitative and quantitative confocal imaging of thick and structurally complex specimens. In particular, the facility will be used to (1) study the interactions of vascular wall cells and components of the extracellular matrix in intact rat blood vessels, as well as in cell culture; (2) examine the localization of cell surface and intracellular proteins in polarized epithelial cells; (3) investigate the assembly of the actin cytoskeleton during differentiation of absorptive epithelial cells; (4) conduct in situ mapping of genes on human chromosomes; (5) study the organization and physiology of rat hepatocytes cultured for extensive periods in collagen gels; (6) localize Mullerian Inhibitory Substance and its receptor during gonadal differentiation; (7) analyze liver parenchymal cell heterogeneity; (8) study signal peptide independent protein secretion; and (9) study the trafficking pathways of transmembrane and PI anchored MHC-1 molecules. To evaluate the advantages of the confocal microscope, cells and tissues used in several of the projects listed here were examined using an instrument identical to the one requested in this proposal. These preliminary data confirm that the optical sectioning, high resolution, and image processing capabilities of the requested instrumentation are essential to successfully carry out the proposed research. The confocal facility will be housed in the Surgery Research Unit at the L.E. Martin Research Building in Charlestown, which is where laboratories of all of the investigators listed in this proposal are located. The instrumentation will be shored by members of the Departments of Surgery, Medicine, and Pathology. It will also be made available to other investigators at the Martin Laboratories, at the discretion of the Internal Advisory Committee, based upon both suitability and the time constraints imposed by the projects already described in this proposal. By having the proposed instrument in an area of active basic and clinical research, we hope to apply confocal microscopy not only to basic biological questions, but also to problems of clinical relevance.
