We have implemented the technique of automated electron tomography in a 300 kV field-emission TEM equipped with a post-column energy-filter to determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles. Series of unfiltered or energy-filtered digital images each containing 2048 x 2048 pixels were acquired using a cooled CCD camera over a specimen tilt range from -70 degrees to +70 degrees and for dual tilt axes rotated by 90 degrees. These image series were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. 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 novel technique for performing 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. We are currently investigating application of the technique to map the distribution of nucleic acid within the cell nucleus.
| 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 |
