Practical Protein Chemistry - A. Darbre 1989

X-ray crystallography and electron microscopy
Electron microscopy
Electron microscope

An Electron Microscope is similar to a Light Microscope in that it incorporates image-forming lenses (Fig. 20.12). Electrons are emitted by the hot cathode filament of the electron gun and accelerated by a high electrical potential difference between the gun and the rest of the microscope. Such a stream of electrons is equivalent to a beam of monochromatic light, and therefore all the theoretical principles underlying light Cell/15.html">Microscopy apply equally to Electron microscopy. In most modern microscopes, the specimen under investigation is mounted on a thin carbon film supported by a copper grid (Fig. 20.13). The specimen holder is designed so that the specimen can be easily moved inside the microscope while remaining completely free from spontaneous drift and vibration during visual observation and photography.

The specimen is placed inside a special device—an anticontamination trap cooled to the Temperature of liquid nitrogen via a copper rod connected to a coolant reservoir. Because of the low temperature, substances evaporating from the specimen under electron bombardment, as well as any contaminants that might enter the microscope, condense On the surface of this device. When the electron beam interacts with molecules, chemically active free radicals can be formed, which react with the specimen and substrate material. In the absence of a low-temperature trap, this leads to the buildup of a contamination layer on the specimen, which degrades resolution and frequently gives rise to artifacts.

Positioned below the specimen is the first group of electromagnetic lenses, functionally equivalent to the objective lens of an optical microscope. Located still further down are projection lenses, which project the image onto a fluorescent screen for direct viewing or onto photographic film to obtain a micrograph.

Changing the focal length of the projection lenses alters the magnification produced by the electron microscope. The magnification can reach up to 250,000x, and it can usually be increased by another factor of three during photographic printing without any loss of structural detail resolution in the micrographs. The focal length of the projection system is regulated by the current flowing through the coils of the magnetic pole pieces of the lenses. For this reason, the electronic devices that are part of the microscope must meet extremely stringent requirements, ensuring not only The ability to vary the current flowing through the lenses, but also to strictly stabilize it at any given level. Otherwise, the magnification would continuously fluctuate.

Focusing of the image is achieved by varying the current flowing through the objective lens, and this must be done after every change in magnification. During normal operation, the microscope is simply switched to the desired magnification (with its electronic systems automatically Setting the required currents in the respective lenses), and then the image is focused by adjusting the objective lens current.