Our knowledge of the functional architecture of normal tissue is gained slowly because tools for studying cell structure have been either very tedious to use (electron microscopy) or ineffective in cells of normal thickness (conventional light microscopy). The aim of this proposal is to acquire a system for studying the 3D functional architecture of cells based on the revolutionary new techniques of confocal light microscopy. This system will be utilized by a core user group of cell, developmental, and neuro-biologists who are engaged in the following major areas of research. 1) a mutational analysis of morphogenetic mutants of C. elegans; 2) the distribution of integrins in developing skeletal muscle membranes; 3) examining the changes of distribution of proteins and membranes involved in excitation contraction coupling resulting from targeted mutations in the ryanodine receptor; 4) correlative light and EM studies of the structure and arrangement of microtubules and MAPS in the mitotic spindle; S) the attachment of actin filaments to the cleavage furrow membrane of dividing cells; 6) studying the recruitment of cytoskeletal proteins by the intracellular bacteria Listeria monocytogenes; 7) the microcircuitry of retinal ganglion cell interconnections. The subjects studied by this core user group are diverse. However, we share the need for a rapid way of studying cell architecture in the 0.1 to 1.0 resolution range, a way that is not rendered useless by tissue thickness in the range 0.1-0.5 mm. We have jointly used a previous generation laser scanning confocal microscope very productively in our research, and have demonstrated that an up-to-date instrument would open up exciting new possibilities for all of us.
