SYSTEM FOR QUANTITATIVE 3D CONFOCAL LIGHT MICROSCOPY

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Our understanding of the functional architecture of the cell and its relationship to both genetic and developmental events has until recently been gained slowly. This is because the tools for studying cell structure have been either tedious to use (electron microscopy) or ineffective in cells due to lack of resolution (conventional light microscopy). The aim of this proposal is to acquire a system for studying the functional 3-D architecture of cells based on a revolutionary new technique of confocal light microscopy. This system will be utilized by a core group of seven cell and developmental biologists who collectively are engaged in seven independent areas of research which focus on the molecular events involved in intracellular organelle communication, assembly and development. These areas are: 1) the mechanism of morphometric gradient generation and its utilization in the developing embryo; 2) the physical basis of zygotic mitochondrial membrane mixing and its genetic consequences; 3) analysis of the mechanism of protein sorting in the polarized cell; 4) analysis of protein folding and its role in protein movement through organelle membranes; 5) screening and characterization of tumorogenesis within different tissues of the transgenic mouse; 6) the physical and genetic analysis of chromatin attachment sites within the cell nucleus; 7) architecture and genetic analysis of the translocation complex mediating transport through the nuclear envelope. The intracellular locations studied by this core user group are diverse; however, we share the need for a rapid and sensitive technique for analysis of cell architecture in the 0.1 to 1.0 micron resolution range in tissues and cells with thicknesses in the range 0.1 to 0.5 mm. Different members of the core group have explored the use of laser scanning confocal microscopy and have found that this newly developed technique makes available new possibilities for analysis which previously were not available.