Perspectives of Molecular and Cellular Electron Tomography

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

“…The analysis of samples by electron tomography was performed on 250-nm-thick sections, as described previously (Koster et al, 1997;Ladinsky et al, 1999). Briefly, 5-or 10-nm gold particles were placed on both surfaces of the plastic sections (prepared as described above).…”

A cell-centered database for electron tomographic data

“…Great progress has been made in data collection (Koster et al, 1997), fiducial tracking (Kremer et al, 1996), and computational speed and parallelization of algorithms (Ferna andez et al, 2002;Smallen et al, 2000), all of which streamline and accelerate the process of tomographic reconstruction to the point where experimenters can go from data collection to reconstruction in less than a day. Analysis of these reconstructions is still far from routine, however, and currently represents the major bottleneck for tomographic studies.…”

“…As more examples of structures such as synapses are added to the repository, additional analyses of structural variability become possible and the relationship of this variability to processes such as synaptic functioning can be modeled. Tomography researchers interested in merging cellular and molecular data will also benefit from having a repository of cell-level structures to search for molecular signatures (Bohm et al, 2000;Koster et al, 1997). In this case, the CCDB can work in concert with molecular databases, e.g., the IIMS database (http:// msd.ebi.ac.uk/iims.html) or the PDB, which can provide templates for pattern searches in cellular images.…”

“…Researchers are employing tomography not only to investigate the structure of purified protein complexes, but also to investigate their structure in situ, fitting the lower resolution tomographic data with atomic models or molecular envelopes obtained by molecular microscopy and X-ray crystallography (McEwen and Frank, 2001;Taylor et al, 1999). Others are using the superior resolution of cell-level tomograms to search for molecular signatures of proteins in complex cellular environments based on an understanding of protein structure, without the need for protein-specific stains (Bohm et al, 2000;Koster et al, 1997).…”

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

“…In addition, some of the advantages of cryopreparation are retained using the technique of freeze-substitution, in which the water in a vitrified frozen specimen is substituted with a solvent at low temperature and the resulting dehydrated cells or tissues are embedded, sectioned and stained in the standard way. Indeed, freeze-substitution has been extensively used in combination with electron tomography (ET) for accurate 3D imaging of cellular ultrastructure at 5-10 nm resolution (McEwen and Marko, 2001;McIntosh, 2001;McIntosh et al, 2005) Nevertheless, it is not generally possible to identify directly specific macromolecular complexes in 3D using ET (Baumeister, 2002;Baumeister, 2005;Koster et al, 1997;McIntosh, 2001;McIntosh et al, 2005). This is because the pattern of stain does not exactly match the shape of the macromolecules to which the heavy atoms bind.…”