The newly developed instrumentation and software now allows three-dimensional reconstruction, in a generalized fashion, of subcellular structures, at both the light and high voltage electron microscopic levels. Eighteen nuclei have been reconstructed and interpreted in a quantitative fashion. Six nuclei have their cytogenetic loci aligned with the three-dimensional structures showing that many chromosome features are three-dimensionally determined. It is proposed to determine, three-dimensionally, many adjacent polytene nuclei in defined developmental stages and tissues so that it will be possible to see in a quantitative fashion the structural relationships. Many computational approaches are proposed to process the images, to refine the structures, to position the bands, hence genes three-dimensionally, and to determine the fine structure of nuclei. These studies will also utilize monoclonal antibodies to localize active gene loci and their close associations one with another and the nuclear envelope, and to relate structure to function. Diploid nuclei will, in addition, be three-dimensionally reconstructed at highest possible light microscope resolution in intact Drosophila embryos. Questions related to differences in the 3D patterns of the interphase chromosomes as a function of development, and the cell cycle will be studied. The laboratory will continue to reconstruct mitotic chromosomes and interband/bands from polytene chromosomes from Drosophila in a resolution-overlaping fashion made possible by the three-dimensional light microscopy and much higher resolution of the high voltage electron microscope utilizing systematic high tilt about 70 degrees for three-dimensional structure. New computational approaches to image processing in three dimensions are being continued. New image collection hardware for the light microscopy and high voltage electron microscopy are proposed.
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