Electron Tomography Of Cellular Structures

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We have implemented the technique of automated electron tomography in a 300 kV field-emission TEM equipped with a post-column energy-filter to determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles. Series of unfiltered or energy-filtered digital images each containing 2048 x 2048 pixels were acquired using a cooled CCD camera over a specimen tilt range from -70 degrees to +70 degrees and for dual tilt axes rotated by 90 degrees. These image series were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) of cultured neurons from rat brain prepared by high-pressure freezing, freeze-substitution and heavy metal staining. The structures of interest associated with the PSDs include CAM kinase II, scaffolding proteins as well as cytoskeletal fibers. To complement studies being conducted by NIDDK investigators to understand defects in the endocytic trafficking pathway that occur in Niemann-Pick Type C Disease, we are determining the cellular ultrastructure of endosomal vesicles and endosomal tubules in cultured fibroblasts and CHO cellls containing wild-type or mutant NPC protein. The reconstructions show multi-lamellar tubules that appear to correlate with late endosomal tubules previously observed in fluorescent light microscopy of labeled cells.