Practical Protein Chemistry - A. Darbre 1989

X-ray Crystallography and Electron Microscopy
Electron Microscopy
Three-Dimensional Electron Microscopy

The images we observe under a Microscope are two-dimensional projections of the three-dimensional Structure OF THE specimen under investigation. By using a goniometer—a device that tilts the specimen within the electron beam—it can be examined from various angles. A series of such images provides significantly more structural information than any single image alone. The most informative approach, however, is to reconstruct the three-dimensional structure of the object based on these images. For certain specimens, this is indeed feasible, although the process is quite time-consuming. The mathematical framework employed here is analogous to that of X-ray crystallography. The micrographs of the studied object, taken at different angles, are converted into numerical form using a densitometer and entered into computer memory. The computer-generated three-dimensional image of the structure can be visually displayed in various ways. Typically, it is presented as a series of sections, similar to those obtained with a microtome for histological samples embedded in a polymer matrix [3, 4].

To date, high-quality three-phase electron density maps have been obtained for several well-ordered "stained" objects. Fig. 20.17 illustrates such a map calculated for ribosome crystals [14]. Such maps are analogous to low-resolution X-Ray Diffraction maps (5 nm).

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FIG. 20.15. An optical diffractometer in which a photomicrograph is illuminated by a parallel beam of light, thereby generating a diffraction pattern. A filter and a lens positioned downstream of the diffraction pattern reconstruct the filtered image.

FIG. 20.16. Optical filtering of a two-layered structure image: a — photomicrograph of an Actin tube; b — diffraction pattern of the photomicrograph containing maxima from both layers and representing a superposition of two reciprocal lattices; c — lattice of diffraction maxima selected for reconstruction and indicated by dashed lines; d — filtered image reconstructed from the selected lattice.

FIG. 20.17. Three-dimensional reconstruction of a ribosome crystal unit Cell based on a series of micrographs taken at different angles. The Ribosomes were found in the crystalline state within oocytes of the lizard Lacerta sicula. L and S denote the large and small ribosomal subunits, respectively.