We have implemented the technique of electron tomography on a digital energy-filtering, 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 images each containing 1024 x 1204 pixels are acquired at between sixty and eighty different angles from -80 degrees to +80 degrees of tilt using a cooled CCD camera. These images are aligned and transferred into a computer program to obtain the three-dimensional reconstruction. We have found that the increase in effective specimen thickness at high tilt angles makes it advantageous to filter the images to remove the effects of chromatic aberration in electron tomography of plastic-embedded sections. However, the advantage of energy filtering is expected to be even greater for analysis of frozen hydrated specimens. We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) labelled for PSD 95, a major scaffolding molecule, and the PSD-associated enzyme CaMKII. The PSDs were isolated from rat brain and were reacted with gold-labeled antibodies. In another application, we have studied virion assembly in BSC-1 cells that were infected with vaccinia virus. Electron tomograms of plastic sections cut to a thickness of 400 nanometers revealed structural information about crescent-shaped and immature virions.
| Aronova, M A; Kim, Y C; Zhang, G et al. (2007) Quantification and thickness correction of EFTEM phosphorus maps. Ultramicroscopy 107:232-44 |
| Leapman, Richard D (2004) Novel techniques in electron microscopy. Curr Opin Neurobiol 14:591-8 |
| Daniell, Sarah J; Kocsis, Eva; Morris, Edward et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol Microbiol 49:301-8 |
