The purpose of this grant proposal is to acquire a Confocal Microscope Imaging System for a broad based group of researchers that utilize light microscopic analysis. This instrument has only recently become commercially available, and provides a significant improvement over conventional microscopes in numerous critical aspects of performance. These performance improvements will allow each member of the group to dramatically increase the resolution of the data that they are obtaining and to address questions not previously considered feasible. Imaging of subcellular structures as well as the organization of cells within tissues has traditionally been accomplished using the standard light microscope with contrasting methods or by epifluorescence microscopy. The confocal microscope has added a new dimension to these standard techniques. The net effect of this system is to eliminate stray light from out of the focal plane, thereby eliminating the flare that is such a problem with conventional epifluorescence images. The net effect is increased resolution, decreased background and increased sensitivity of detection. The confocal microscope is also able to optically section a fairly thick specimen. When coupled with a computer system and appropriate software, the data from a series of optical sections can be reassembled by the computer to form a 3-dimensional reconstruction of the fluorescent emission pattern from the specimen. This can be used to localize subcellular structure or protein within a cell, or a particular labelled cell within a thick tissue. This capability is unique to the confocal microscope. The group of investigators who constitute the major users group for this grant application are all involved in research that relies heavily upon microscopy in general and fluorescence microscopy in particular. All are involved in determining the precise 3-dimensional organization of cells and sub-cellular structures using fluorescent antibody techniques. One member is active in investigating the cytoskeleton and interactions of the cytoskeletal components necessary for motility, cytokinesis and morphogenesis. The other three are investigating the organization, structure and function of the nervous system and neuromuscular interactions. In each case the information obtained is limited primarily by the optical properties of the microscopes we use. With the confocal microscope, both the level of resolution of our data will be increased and new experimental approaches will be possible.
