Practical Protein Chemistry - A. Darbre 1989

X-ray crystallography and electron microscopy
Electron microscopy
Resolution and limitations of the method

In Cell/15.html">Microscopy, resolution depends on the wavelength of the radiation used. In a Light Microscope, at a wavelength of 500 nm, the resolution does not exceed 250 nm. The Use of X-Ray Diffraction and computer-assisted Image Processing makes it possible to achieve a resolution of 0.1 nm at a radiation wavelength of 0.15 nm. In an Electron microscope, at an accelerating voltage of 100 kV, the electron wavelength is ~0.003 nm. However, for technical reasons, the actual resolution of these instruments reaches only ~0.3 nm, which in principle already allows many details of molecular Structure to be distinguished. Unfortunately, in the case of biological Materials, it is extremely difficult to prepare samples for examination while preserving their structure intact at such resolutions. The limitation in resolution is caused not by the microscope itself, but by current Sample preparation Methods for microscopy, which make it possible to preserve the structure under investigation in a vacuum under electron beam bombardment.

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FIG. 20.12. Schematic diagram of the main lenses in an electron microscope. Unlike an optical microscope, in an electron microscope the radiation source is located at the top, and the magnified image is formed at the bottom.

The traditional way to solve this problem is "staining" structures with heavy metals. Therefore, the resulting image typically represents the distribution pattern of the staining agent within the sample, from which the Features of the object's molecular structure can be inferred. Resolution in these cases generally does not exceed 1.5 nm. To improve resolution down to 0.6 nm, new methods for Molecular Microscopy have been developed relatively recently. In these methods, an increase in the signal-to-noise ratio is achieved, as in the case of X-ray diffraction, by using ordered molecular assemblies—crystals. Below, the principles of routine Electron microscopy methods will be discussed, followed by their latest advancements.

FIG. 20.13. In an electron microscope, the sample is placed on a carbon film supported by a copper grid (copper provides good electrical and thermal contact between the specimen and the sample holder). The upper part of the figure illustrates the negative staining method.