Electron Tomography Of Cellular Structures

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We have implemented the technique of electron tomography in a digital transmission electron microscope equipped with a post-column energy-filter in order to determine the three-dimensional arrangement and structure of supramolecular assemblies and subcellular cellular organelles. Series of energy-filtered images each containing 1024 x 1204 pixels are acquired at between sixty and eighty different tilt angles from -80 degrees to +80 degrees using a cooled CCD camera. These image series are analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. Gold particles are adsorbed onto the sections to facilitate alignment of the images in each series. 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 extend the capabilities of electron tomography, we have developed a new technique enabling three-dimensional microanalysis based on core-excitation images. Using this approach, we have shown that it is possible to map the phosphorus in ribosomes in high-pressure frozen and freeze-substituted preparations of C. elegans. The method can distinguish between protein and nucleic acid in unstained specimen and might therefore find applications in the study of the cell nucleus.