Electron Tomography Of Cellular Structures

Office Of The Director, Nih

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Abstract

To determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles, automated electron tomography has been successfully implemented in a 300 kV field-emission transmission electron microscope (TEM) equipped with a post-column energy-filter. Various specimen holders have been evaluated for acquisition of dual-axis tilt series over an angular range of -70 to +70 degrees in order to test goniometer eucentricity. Digital images each containing 2048 x 2048 pixels were collected by means of a quadrant read-out cooled CCD camera, and were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. Computer programs capable of providing both surface and volume rendering were used to visualize structures of interest. We have applied electron tomography to a number of systems ranging from simple prokaryotic cells to complex tissues. For example, we have determined the three-dimensional structure of the bacterium Spiroplasma in order to investigate its cytoskeletal ribbon, which enables motility. We have also recorded tomograms from post-synaptic densities (PSDs) in high-pressure frozen cultured rat hippocampal neurons to investigate changes in the arrangement of CAM kinase II, PSD95, and associated scaffolding proteins under stimulated and unstimulated conditions.