The confocal laser scanning microscope rejections out-of-focus light and allows the visualization of internal cellular structures at a level of quality not attainable by conventional light microscopes. The most recent generation of confocal microscopes offers substantial improvements in terms of ease of use, digital image processing, and software control of instrument operation. The Department of Cell Biology at Harvard Medical School comprises 29 laboratories, but does not possess a confocal microscope; the only available instrument is a 6 year old MRC600 in the Department of Neurobiology that is heavily oversubscribed because of a lack of alternatives. The Department wishes to obtain a Zeiss LMS410, which was chosen after on-site demonstrations by the leading manufacturers of confocal microscopes. While the instrument will be used by many members of the Department, a core user group has been identified. Brief descriptions of selected projects are given here; complete descriptions are given in the body of the proposal. (1) Testing the fixed-cortex hypothesis in mesenchymal cells migrating in three-dimensional matrices. Caged Resorufin-actin will be microinjected into avian mesenchymal cells, and specific patches of cortical actin will be photoactivated with 365 nm light from an Argon ion laser. The behavior of the fluorescent patch will be followed by time- lapse confocal microscope while the cells are migrating in collagen gels. (2) Distribution of mRNA's for growth factors and growth factor receptors in amphibian and avian embryos. RNA fluorescence in situ hybridization will be used to localize specific mRNA species in fixed whole-mount embryos. (3) Cell migrations and lineage analysis in the Xenopus embryo. Zygotes will be loaded with photoactivable caged Fluoresceindextran, and small groups of cells in the tailbud will be activated with 365 nm light from an Argon ion laser. Lineage analysis will be performed by time-lapse 3 dimensional confocal microscopy of photoactivated fluorescein. (4) Co- localization of pathogens in the endocytic compartment of thick mammalian cells. High-resolution 3-D image reconstruction of immunostained specimens will be used to study the trafficking of reovirus and Salmonella typhimurium in the endocytic compartment of mouse macrophages.