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.
| 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 |
