This revised proposal requests funding for a laser scanning confocal microscope (LSCM) and appropriate accessories. The applicants are nine faculty members from the Departments of Anatomy, Growth and Development, Pharmaceutical Chemistry, Pharmacy, Physiology, and Stomatology (spanning the Schools of Dentistry, Medicine and Pharmacy). The research programs of the investigators require detailed information on the three-dimensional localization of molecules in extracellular and intracellular space. Projects are described which involve applications of LSCM both to whole tissue samples and to cellular and subcellular events. Specifically, the following areas of significance will be explored: epidermal barrier development and enhancement of percutaneous transport, migratory and invasive activity of early embryonic cells, tumor cell invasion of basement membrane and interstitial matrices; receptor-mediated endocytosis and the uptake of antigenic peptides, biochemical and biological characterization of transforming growth factors (TGF-alpha and TGF-beta), protein traffic in polarized epithelial cells, the regulatory mechanism(s) of neuronal nicotinic acetylcholine receptors, and the uptake and processing of oligonucleotides by cells in culture. For each research program outlined, the ability of the LSCM to analyze and quantify the spatial distribution of molecules through membranes, and within and around cells, is crucial. This quality of sophisticated information cannot be achieved with conventional immunofluorescence microscopy, nor can it be accurately assessed using electron microscopy. Considerable augmentation of multiple research directions will result, therefore, from the availability of a confocal microscope at this time. Although there are such instruments at UCSF, access for the present investigative team is very restricted and is becoming progressively more difficult. The School of Pharmacy administration has committed space for the location of a new apparatus in the event that this application is successful. We believe, therefore, that we have an important need for this shared equipment, that feasible alternatives are not available at our institution, and that a strong Departmental and School commitment to the long-term operation and maintenance of the LSCM is in place. The instrument requested is a BioRad- 600 laser-scanning confocal microscope. The overall system will consist of several interconnected components: confocal imaging system, standard epifluorescence microscope, video printer, and optical disk for mass storage.
