Electron Tomography Of Cellular Structures

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Abstract

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.