Electron Tomography

 Electron tomography is defined as the generation of a 3-D reconstruction from an electron microscope tilt series (1). This powerful technique is closely related to computerized axial tomography used by CAT scanners in radiological imaging in the computational methods used to calculate a 3-D structure from many two-dimensional images or projections recorded over a wide range of tilt angles. The use of tomography has proved valuable not only for the determination of macromolecular complexes (2-4), but also for the visualization and analysis of relatively large, complex biological structures. Because of variable size, large-scale biological structures such as organelles elude crystallographic or single-particle approaches that require multiple images of identical structures. Electron tomography is especially powerful for these complex structures, but until recently, it has not enjoyed widespread application because of obstacles that are now being removed (5). Electron tomography presently is the imaging technique that provides the highest 3-D resolution (capable of 5–10 nm) of the internal features of organelle-size structures. This is accomplished by recording a series of images of a single specimen over a wide range of tilt angles with a small interval of tilt angle. Typically such a tilt series would consist of 61 images recorded at tilt angles from –60° to +60° in 2° increments. The individual images must be digitized (unless recorded directly in digital form using a CCD camera attached to the microscope), aligned to a common origin, and processed using computer algorithms, similar to those used in computerized tomography in medical imaging, to reconstruct a 3-D volume from a series of 2-D projections. Since whole cells and organelles, such as mitochondria, are relatively large, the images must be recorded from specimens embedded in semithick sections, 0.25–1 µm thick. Such thick specimens require the use of higher voltage electron microscopes, intermediate voltage electron microscopes (IVEM's( with accelerating voltages up to 400,000 volts, or high-voltage electron microscopes with accelerating voltages up to 1,200,000 volts. By comparison, typical transmission electron microscopes have accelerating voltages of approximately 100,000 volts. The higher voltage electron microscopes are relatively expensive, and most users wishing to do electron tomography of thick specimens must use instruments made available in national facilities.

 Until recently, the bulk of our knowledge of the architecture of organelles and bacteria has come from untilted images of sections. The 3-D architecture is usually inferred from these images, sometimes with the aid of stereo imaging or serial-section reconstruction. Although thin-section electron microscopy provides relatively high-resolution images, an incorrect impression of the 3-D structure may be obtained because one is looking at only a very thin slice through a complex 3-D object. On the other hand, electron tomography offers an opportunity to map topology more accurately by providing improved resolution along the z-axis, while circumventing major deficiencies in stereo imaging and serial-section reconstructions. This technique provided the resolution necessary to observe characteristics of mitochondrial structure that differ from long-held conceptions (6,7). These newly appreciated characteristics include: (i) All observed cristae connect to the inner boundary membrane via narrow, tubular openings, termed crista junctions. (ii) Tubular cristae merge, sometimes from opposite ends of the mitochondrial periphery, to form lamellar compartments. (iii) Contact sites are not clustered about crista junctions. The full power of electron tomography is now being explored to generate 3-D distributions of specifically labeled or immunolocalized components.

References

1. J. Frank (1992) Electron Tomography, Plenum Press, New York. 

2. R. A. Horowitz et al. (1994) J. Cell Biol. 125, 1–10. 

3. H. Mehlin, B. Daneholt, and U. Skoglund (1992) Cell 69, 605–613.

4. M. Moritz et al. (1995) Nature 378, 638–640. 

5. J. Frank (1995) Curr. Op. Struct. Biol. 7, 266–272.