During embryogenesis the body plan of an organism is determined, tissues are demarcated, and organ systems formed. Because of its limited cell number, transparency, and invariant cell lineage, C. elegans provides an excellent model system for the detailed analysis of embryogenesis. Until now, however, there have been no computer-assisted systems available for the reconstruction and motion analysis of an entire embryo (i.e., including every cell, nucleus, and cytoplasmic domain) from fertilization to the end of cleavage. The methods that have been available have been for the most part two dimensional and devoid of motion analysis software. In recent years, we have developed computer assisted systems (3D-DIAS) for reconstructing and motion analyzing in 3D the surface, nucleus, and pseudopodia of individual crawling cells (1,2). Using DIC optics and stepper motors, cells are optically sectioned in a two-second period. Optical sections are digitized and the edges of the cell, nucleus, and pseudopodia are identified by automatic and manual methods. The outlines are converted to b -spline models that are stacked and used to construct faceted 3D images of a crawling cell every two seconds. The series of 3D reconstructions is converted into a 3D computer movie that can be viewed at any angle through a stereo workstation. Every component of the dynamic 3D image (cell surface, pseudopod, nucleus) can then be individually reconstructed in 3D over time, and motion analyzed in 3D, allowing the assessment of high-resolution behavioral phenotypes. 3D-DIAS has been used to elucidate the mechanism of chemotaxis and the roles of a number of regulatory and cytoskeletal components in Dictyostelium discoideum (e.g., 3). We will present here for the first time a new computer-assisted system, 3D-DIAS emb , that we have developed to reconstruct and motion analyze developing embryos in three-dimensions. This system reconstructs the surface of every cell and nucleus in 3D. C. elegans embryogenesis was used as the initial model to develop this system. 60 optical sections are collected through the embryo in two seconds using differential interference contrast microscopy, and the process repeated every 5 seconds. The optical sections are stacked and converted to 3D faceted reconstructions of every cell and nucleus through embryogenesis. The audience will be provided with red and blue glasses in order to view 3D reconstructions of wild-type embryos from 1-50 cells, in which each cell and each nucleus is converted into a 3D faceted image. This system allows the user to view a developing embryo from any angle through time, to remove any cell or nucleus at any time and analyze its behavior and lineage (i.e., every morphometric change, every nuclear division, every association with other cells over time), and color code cells and nuclei in any desired manner. Cell surfaces can be subtracted and only nuclear divisions reconstructed and motion analyzed over time. The system also provides vector flow plots that reflect cytoplasmic flow at any depth and in any desired cell over time. The power of the technology for analyzing mutant phenotypes will be demonstrated by comparing wild-type and
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