Electron Tomography

Abstract

Electron tomography (ET) is an emerging electron microscopy (EM) technique for the three‐dimensional (3D) visualization of the cellular architecture and molecular organization in native cells at nanometer scale. ET thus bridges the gap between the low‐resolution imaging techniques and the high‐resolution structural techniques. In ET a series of images is taken from a single specimen at different projecting orientations. The 3D map computed from these images is subsequently subjected to visualization and interpretation. Thanks to several recent innovations in EM, including improved specimen preparation techniques, novel EM hardware and software, reliable genetic and molecular labelling approaches, ET is rapidly becoming a powerful bioimaging tool for precisely dissecting the macromolecular organizations and cellular events captured from living cells in health and disease. Thus many previously unanswered molecular mechanisms responsible for the specific cellular functions could be clearly elucidated by the ET technology.

Key concepts:

  • Electron tomography allows visualization of cellular architecture and molecular organization in native cells at nanometer scale in 3D.

  • Electron tomography bridges the resolution gap between the low resolution imaging techniques and the high‐resolution structural techniques.

  • The biological specimen has to be specially prepared before it is imaged in the electron microscope.

  • Data acquisition strategies have to be used to obtain reasonable image contrast and reduce the electron‐dose damage.

  • The 3D map is calculated from a series of 2D EM projection images recorded from the specimen at different projecting orientations.

  • Several post‐processing stages are intended to facilitate the interpretation of the complex 3D maps.

Keywords: electron tomography; cryo‐electron microscopy; cell biology; bioimaging

Figure 1.

Comparisons of 3D bioimaging tools.

Figure 2.

Projection acquisition and tomographic reconstruction. (a) Projection. (Left) Tilt series are acquired by tilting the specimen around a tilt axis (here perpendicular to the sheet) at small increments over a limited tilt range. (Centre) Projection process of an object made up of four dots with different radii at 0o tilt. (Right) Acquisition of several projections of a tilt series from the object. (b) Assembling the Fourier transform (FT) of the three projections at −45°, 0° and 45° ((a), right) in the Fourier space, which is progressively filled as more projections are acquired and their FTs assembled. Afterwards, an inverse FT yields the reconstructed object. The ‘missing wedge’ is marked with grey triangles. (c) The reconstructed object under ideal conditions (sampling and covering). (d) Reconstruction of the object from the tilt series with back‐projection. From left to right: the reconstruction with tilt series over the range [−50°,+50°] with 1, 3, 5, 15 and 25 projections. The influence of the limited tilt range and the angular sampling are apparent.

Figure 3.

Specimen preparation techniques for electron tomography.

Figure 4.

The ultrastructures of cells preserved by different specimen preparation techniques. (a) and (b) Well‐preserved cellular organization and ultrastructures observed in ileal epithelial cells in suckling rats prepared with HPF/FSF – microtubules (pointed by red arrows) attached to tubular vesicles; ordered structures on the inner surface of large vesicles (pointed by blue arrow); membranes of vesicles and mitochondria are well‐preserved; (unpublished results) (c) Molecular organizations of desmosomes and cell skeletons observed in mouse skin prepared with HPF/FSF (reproduced from He et al., with permission from the American Association for the Advancement of Science). (d) Vesicle with ordered structures observed in ileum of suckling rat prepared with HPF/FSF (unpublished result). (e) Tracing endocytosed 1.4 nm Nanogold particles by HPF/FSF‐gold‐enlarging techniques (He et al., ) (unpublished result). (f) Coated vesicles in thin region of fibroblast prepared by plunge freezing (reproduced from Koning et al. with permission from Elsevier). (g) High‐resolution ultrastructures of desmosomes in human skin prepared by frozen‐hydrated sectioning (reproduced with permission from Al‐Amoudi et al.).

Figure 5.

Interpretation of tomograms. (a) Vaccinia virus (top) denoised with anisotropic nonlinear diffusion (bottom; Cyrklaff et al., ). (b) Cellular context of tubular vesicles (right) modelled by manual segmentation (left; reproduced from He et al. with permission from Nature Publishing Group). (c) Atlas of the 80S ribosome (bottom) built from a section of S. cerevisiae (top) after detection with template matching, alignment and averaging (Pierson et al., ).

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How to Cite close
He, Wanzhong, and Fernández, José‐Jesús(Jan 2010) Electron Tomography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021877]