The purpose of this grant application is to obtain funds for a Zeiss Laser Scan Microscope (LSM) equipped with a Zeiss VIDAS Image Processing System. The LSM provides both confocal optics for fluorescence analysis and laser scan transmitted capabilities for phase contrast and DIC observations. This item of equipment will be used by three PHS extramural awardees who are all members of the Department of Cell Biology and Anatomy at Northwestern University Medical School. All three of these primary users of the LSM currently require extensive use of standard immunofluorescence microscopy for many of the specific aims of their funded projects. The confocal mode of the LSM is designed to optimize resolution of immunofluorescence preparations with the highest numerical aperture infinity corrected lenses. It achieves this through the use of a laser scanning system capable of resolving thin focal planes within microscopic specimens of varying thickness including single cells in culture, as well as in cells in aggregates and in tissue sections. This focussing achievement in turn permits users to carry out 3-dimensional reconstructions from multiple sequential images obtained through the use of a stage equipped with an integrated Z-scanner motor drive. These images are stored digitally and 3-D reconstructions are achieved with the VIDAS system. These 3-D analyses are impossible with conventional fluorescence optics. The projects of the three major investigators are: (1) Determination of the structure and function of intermediate filament systems (Goldman). (2) The regulation of the organization and function of cytoskeletal elements in Dictyostelium discoideum (Fukui). (3) The structure, function and assembly of hemidesmosomes and desmosomes in mammalian cells and tissues (Jones). In this proposal we emphasize the benefits of the LSM for all these investigators. The LSM is a relatively new research tool which will revolutionize many areas of structural biology, especially those dealing with subcellular structure and function.
