We have developed a new method for the three-dimensional reconstruction of cellular structure in order to understand this structure at a macromolecular level and how structure is related to biochemical and biological function. We use electron micrographs produced by a high voltage electron microscope (a national resource sponsored by NIH) in Boulder, Colorado and other electron and light micrographs taken at NIH. This method was extended to a microcomputer with high resolution color graphics. Software for the image digitization, alignment-of-sections and for the whole image reconstruction from the separate sections has been accomplished. We continue now to develop software for realtime rotation and image representation in three- and two-dimensions. This methodology will be employed to determine the organization of microtubule nucleation centers in cells. It will be important for studies of the determinants of cell shape, and development. Cytoplasmic structure and diffusion within cells will be also analyzed. In addition, studies of embryonic, and also brain development are planned using this newly constructed facility. The second part of this project is concerned with volume, surface area and space for diffusion of the cytoplasmic matrix. We developed a new image analysis method to measure the volume fraction and the surface area occupied by cells by the gelatinous cytoplasmic matrix (the cytoskeleton and microtrabecular lattice). It involves analysis of electron-microscopic data using a video frame buffer (DCRT's Evan and Sutherland System). The results obtained so far show that these structures occupy no more than 10-30% of the cytoplasmic volume. Comparing it with protein diffusion results yielded values for binding energies of proteins to the cytoplasmic matrix. A study on the effect of osmotic conditions on the volume of the cytoplasmic matrix has been initiated and will be further studied. The use of these techniques will be extended to study immunocytochemical systems.