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Resource for the Visualization of Biological Complexity (RVBC)

Electron microscopic tomography

Electron microscopic tomography is used to obtain 3D ultrastructural data on biological specimens that are too large and variable for the single-particle averaging approach, and too complex for viewing in stereo (Frank, 1992; Frank et al., 2002; McEwen and Marko, 2001). In tomography, images are recorded from a specimen tilted at many different angles, often around two orthogonal axes (Penczek et al., 1995), and combined computationally into a 3-D reconstruction. The z-axis (depth) resolution within the final reconstruction depends on the number of tilt images and the thickness of the specimen, and is generally in the range of 4-10 nm. This is superior to the z-axis resolution obtained in 3D reconstructions from serial thin sections, which is limited by the section thickness (usually 50-80 nm). Since the early 1990s, electron tomography has been applied routinely to 0.2 - 1.0 μm thick sections of plastic-embedded specimens, using the IVEM or HVEM. Recently, tomographic analysis of more native (albeit more labile) frozen-hydrated specimens, free of fixatives and stains, has been made possible by implementation of low-dose, automated digital recording (Rath et al., 1997). Tomography of frozen-hydrated specimens is carried out on the IVEM and the F20. Careful selection of imaging conditions is important for frozen-hydrated specimens (McEwen at al, 2002), and the IVEM also has an energy filter, which enhances contrast for thicker specimens. The energy filter also provides prospects for combining tomography with elemental analysis (Marko, et al., 2004). The software used in electron tomography is described on the image processing page.

References

Frank, J. (ed) (1992) Electron tomography. Plenum, New York.

Frank, J., Wagenknecht, T., McEwen, B., Marko, M., Hsieh, C., and Mannella, C.A. (2002). Three-dimensional imaging of biological complexity. J. Struct. Biol. 138:85-91.

Marko, M., Wagenknecht, T., Mannella, C. (2003). Correlative electron tomography and elemental microanalysis in biology: a preview. Microscopy Today 11(6):14-20.

McEwen, B. and Marko, M. (2001) The emergence of electron tomography as an important tool for investigating cellular ultrastructure. J. Histochem. Cytochem. 49:553-563.

McEwen, B. F., Marko, M., Hsieh, C.-E., and Mannella, C. (2002). Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryo-electron tomography. J. Struct. Biol. 138:47-57.

Penczek, P., Marko, M., Buttle, K., Frank, J. (1995). Double-tilt electron tomography. Ultramicroscopy 60: 393-410.

Rath, B.K., Marko, M., Radermacher, M., Frank, J. (1997). Low-dose automated electron tomography: a recent implementation. J. Structural Biol. 120: 210-218.