(Supported in part by GMS 40198 to C. Rieder and by NSF MCB9420772 to B. McEwen) This project required that methods be developed to tomographically reconstruct small regions of cells followed in the living state by video light microscopy. The general goal was to evaluate several hypothesis concerning how chromosomes move and how the kinetochore/centromere complex on each is assembled. First we used video microscopy to follow a cell in the living state through fixation. Next the embedded cell was serially thick sectioned and the section containing the kinetochore of interest was photographed in a tilt series on the IVEM. The resultant tomogram was then computed and visualized using the SPIDER/WEB image processing software. Different components of the reconstruction (e.g., microtubules and the kinetochore outer plate) were traced using Sterecon. Sterecon also enabled us to make quantitative measurements of microtubule penetration into the outer plate. For illustrations, c omplex surface-rendered and filled-contour models were made with the parallel-processor, reality engine-equipped SGI Onyx computer. During 1997 a large number of tomographic reconstructions were made and analyzed, and parts of this study were published. McEwen, B.F. and A. B. Heagle. (1997) Electron microscopic tomography: A tool for probing the structure and function of subcellular components. Internat. J. Imaging Systems. Technology, 8:175-187. McEwen, B.F., A. B. Heagle and C.L. Rieder. (1997) Probing the mechanisms underlying kinetochore behavior in vertebrate cells using combinations of advanced light and 3-D electron microscopy. Microsc. Microanalysis 3(suppl 2):217-218.
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