Electron Microscopy

electron microscopy

Technique that allows examination of samples too small to be seen with a light microscope. Electron beams have much smaller wavelengths than visible light and hence higher resolving power. To make them more observable, samples may be coated with metal atoms. Because electrons cannot travel very far in air, the electron beam and the sample must be kept in a vacuum. Two different instruments are used. In the scanning electron microscope, a moving beam of electrons is scanned across a sample; electrons scattered by the object are focused by magnetic “lenses” to produce an image of the object's surface similar to an image on a television screen. The images appear three-dimensional; they may be of small organisms or their parts, of molecules such as DNA, or even of large individual atoms (e.g., uranium, thorium). In the transmission electron microscope, the electron beam passes through a very thin, carefully prepared sample and is focused onto a screen or photographic plate to visualize the interior structure of such specimens as cells and tissues.

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Electron Microscopy 

the aggregate of methods in which electron microscopes are used to investigate the microstructure of objects down to the atomic or molecular level, the local composition of objects, and the microfields of objects. Microfields are electric or magnetic fields that are localized at the surfaces of objects or in microscopic volumes within objects.

In the USSR, electron microscopy also is an independent scientific discipline whose subjects and objectives include the development of new electron microscopes and microscope accessories, the improvement of existing instruments, the development of techniques for preparing specimens investigated in electron microscopes, the study of formation mechanisms of electron-optical images, and the development of methods of analyzing the various types of information—not only images—obtained by means of electron microscopes. In Soviet practice, electron microscopy also deals with the development or improvement of other particle-beam microscopes, such as the proton microscope.

The objects investigated in electron microscopy are mainly solids. The specimens studied in transmission electron microscopes (TEM’s), in which electrons with energies of 1 kiloelectron volt to 5 megaelectron volts pass through an object, include thin films, foils, and sections with a thickness ranging from 1 nanometer to 10 micrometers (µ,m), or from 10 angstroms (Å) to 10⁵ Å. The surface and underlying structure of objects substantially thicker than 1 µm is investigated by means of nontransmission types of electron microscopes, such as scanning electron microscopes (SEM’s), electron mirror microscopes, or field-emission microscopes; field-ion microscopes may also be used for this purpose. Powders, microcrystals, aerosol particles, or other particles deposited on substrates may be studied. In TEM’s, the substrates are thin films; in SEM’s, thick substrates are used.

The surface geometrical structure of a thick object may also be studied by means of a replica, that is, an impression of the object’s surface in the form of a thin film of, for example, carbon, a colloid, or Formvar. A replica reproduces surface topography and is examined in a TEM. Before examination, a layer of a metal that strongly scatters electrons—for example, Pt—is usually deposited on the replica in a vacuum at an oblique angle, that is, at a small angle to the replica’s surface. The metal layer shadows the projections and depressions of the surface topography.

The technique of shadow casting is used to study not only the geometrical structure of surfaces but also accumulations of point defects (seeDEFECTS, CRYSTAL), stages of crystal growth, domain structure (seeDOMAINS), and microfields caused by the presence of dislocations. In shadow casting, a very thin layer of shadow-casting particles is deposited on the surface of a specimen and a replica that includes the shadow-casting particles, which are deposited mostly in areas where microfields are concentrated, is then made. The shadow-casting particles may be atoms of Au, Pt, or another element or molecules of a semiconductor or a dielectric.

The accessories of a TEM or a SEM include special small gas-filled chambers that make it possible to study liquid or gaseous specimens that are unstable in a high vacuum, for example, wet biological specimens. Since the radiation damage caused by the irradiating electron beam is fairly high, a mode of electron microscope operation that ensures a minimum radiation dosage must be carefully chosen when biological, semiconductor, polymer, or certain other types of specimens are investigated.

In addition to the investigation of static objects, which do not vary in time, electron microscopy makes it possible to study various processes in their dynamical evolution. Such processes, which are investigated in situ, include the growth of thin films, the deformation of crystals subjected to variable loads, and structural changes that result from electron or ion bombardment.

Owing to the short persistence of the electron, methods of stroboscopic electron microscopy may be used to investigate processes that are periodic in time, for example, the magnetization reversal of magnetic thin films, the reversal of polarization of ferroelectrics, or the propagation of ultrasonic waves. In such methods, an electron beam illuminates a specimen by means of pulses that are in synchronism with the voltage pulses supplied to the specimen. The synchronism ensures that a specific phase of a process is recorded on the microscope display in exactly the same way as in conventional optical stroboscopic instruments. In principle, the ultimate time resolution in stroboscopic electron microscopy is about 10^–15 sec for a TEM. In practice, a resolution of ~10^–10 sec is obtained for both the TEM and the SEM.

Amorphous solids and other solids whose particles are smaller than the resolvable distance for an electron microscope diffusely scatter electrons. To interpret the images of such solids, very simple methods of amplitude electron microscopy are used. For example, in a TEM, the image contrast—that is, the difference in image brightness between adjacent regions of an object—is, in the first approximation, proportional to the difference in thickness between the regions.

The scattering of particles by crystalline solids having regular structures gives rise to the diffraction of the particles. Methods of phase-contrast electron microscopy are used to compute the image contrast for such solids and to solve the inverse problem, that is, to calculate the structure of an object from an observed image. Phase-contrast electron microscopy entails the solution of the problem of electron wave diffraction (seeDE BROGUE WAVES) by a crystal lattice. In solving the problem, inelastic interactions of electrons and an object are also taken into account; such interactions include scattering by plasmons and scattering by phonons.

In high-resolution TEM’s and scanning transmission electron microscopes (STEM’s), images of individual molecules or atoms of heavy elements are obtained. By using the methods of phase-contrast electron microscopy, the three-dimensional structure of crystals or of biological macromolecules is reconstructed from images. In particular, holographic methods are used to solve such problems, and the calculations are performed by means of computers.

A variety of phase-contrast electron microscopy is interference electron microscopy, which is analogous to optical interferometry (seeINTERFEROMETER). In this case, the electron beam is split by means of electron prisms. The specimen, which alters the phase of the electron wave passing through it, is placed in one of the arms of an interferometer. The method may be used to measure, for example, the intrinsic electric potential of a specimen.

In Lorentz electron microscopy, phenomena caused by the Lorentz force are studied. The method is used to investigate either intrinsic magnetic and electric fields or externally applied scattering fields. The fields examined include the fields of magnetic domains in thin films, the fields of ferroelectric domains (seeDOMAINS), and the fields of magnetic read/write heads.

The composition of objects is investigated by microdiffraction methods, that is, by electron diffraction analysis of local areas of an object, by electron probe microanalysis (seeSPECTROCHEMICAL ANALYSIS, X-RAY), or by cathodoluminescence microanalysis (seeCATHODE LUMINESCENCE). Either a characteristic X-ray spectrum or cathodoluminescence is excited when a specimen is irradiated by a finely focused electron beam, or an electron probe; the probe diameter is less than 1 u.m. In electron probe microanalysis, characteristic X-ray spectra are recorded. In cathodoluminescence microanalysis, the cathodoluminescence is detected. Microdiffraction methods are also used to study the energy spectra of secondary electrons, which are emitted from the surface or the bulk of a specimen as a result of bombardment by the primary electron beam.

Much work is being done on the development of methods of quantitative electron microscopy. Such methods entail the precise measurement of various parameters of a specimen or of a process being investigated. Examples include the measurement of local electric potentials, local magnetic fields, and the microgeometry of surface topography.

Electron microscopes are also used for industrial purposes, for example, to fabricate microcircuits by means of photolithography.

REFERENCES

Hawkes, P. Elektronnaia optika i elektronnaia mikroskopiia. Moscow, 1974. (Translated from English.)
Stoianova, I. G., and I. F. Anaskin. Fizicheskie osnovy metodov prosvechivaiushchei elektronnoi mikroskopii. Moscow, 1972.
Utevskii, L. M. Difraktsionnaia elektronnaia mikroskopiia v metallovedenii. Moscow, 1973.
Elektronnaia mikroskopiia tonkikh kristallov. Moscow, 1968. (Translated from English.)
Spivak, G. V., G. V. Saparin, and M. V. Bykov. “Rastrovaia elektronnaia mikroskopiia.” Uspekhi fizicheskikh nauk, 1969, vol. 99, issue 4.
Vainshtein, B. K. “Vosstanovlenie prostranstvennoi struktury biologicheskikh ob”ektov po elektronnym mikrofotografiiam.” Izv. AN SSSR: Seriiafizicheskaia, 1972, vol. 36, no. 9.
Quantitative Scanning Electron Microscopy. London-New York-San Francisco, 1974.

A. E. LUK’IANOV

Biological applications. The use of electron microscopy in biology has made it possible to study the ultrastructure of cells and of extracellular components of tissues. For biological objects, the maximum resolution of electron microscopes is 12–6 A; the magnification may be as high as 800,000–1,200,000. Beginning in the 1940’s, results obtained with electron microscopes have been used to describe the fine structure of membranes, mitochondria, ribosomes, and other cell structures, as well as extracellular structures. In addition, certain macromolecules, such as DNA, have been revealed. Scanning electron microscopy makes it possible to study the surface ultrastructure of cells and of tissue structures not only in fixed objects but also in living animals with a hard chitinous shell, for example, a number of arthropods.
The techniques of preparing biological specimens for electron microscopy include procedures that preserve tissues under conditions of a high vacuum and electron-beam irradiation and procedures that yield a high microscope resolution. Objects are usually fixed by means of chemical reagents, such as aldehydes or osmium tetroxide. They are dehydrated by means of alcohol or acetone and are embedded in epoxy resins. They are then cut into ultrathin sections by means of special microtomes; the thickness of the sections is 100–600 Å. To enhance image contrast, cells are treated with electron stains, which strongly scatter electrons. Such stains include uranyl acetate and lead hydroxide.
To reduce the harmful effects of fixatives on tissues, a tissue specimen may be frozen and then dehydrated at a low temperature by means of acetone or alcohol. Methods that preclude the harmful effects of fixatives on cells are sometimes used. Such methods include lyophilization, or freeze-drying, in which a tissue specimen is rapidly cooled to – 150° or – 196°C and is dehydrated in a high vacuum at a low temperature. A promising method is freeze-fracturing, in which a carbon-platinum replica containing a piece of a frozen object is obtained. Freeze-fracturing has led to substantial changes in concepts about the structure of cell membranes.
Negative staining of specimens is used to study the structure of biological macromolecules or of individual organelles. In this case, the objects being studied appear as light elements on a dark background. The objects appear light because they are transparent to electrons.
The images of molecules obtained in electron microscopes may be analyzed by using methods based on the diffraction of light. The use of high-voltage (up to 3 megavolts) electron microscopes makes it possible to obtain information about the three dimensional structure of cells.
When living arthropods are prepared for investigation, they are incapacitated by ether or chloroform in doses that do not subsequently kill the animals and are placed in the vacuum chamber of an electron microscope. Methods of cytochemistry, including autoradiography, are widely used in present-day electron microscopy.
The use of electron microscopy in biology has substantially changed and deepened previous concepts about the fine structure of cells.

REFERENCES

Kiselev, N. A. Elektronnaia mikroskopiia biologicheskikh makromolekul. Moscow, 1965.
Elektronno-mikroskopicheskaia anatomiia. Moscow, 1967. (Translated from English.)
Balashov, Iu. S., and N. E. Mikkau. ’Izuchenie zhivykh zhivotnykh v rastrovom elektronnom mikroskope.” Priroda, 1977, no. 1.
Tribe, M. A., M. R. Eraut, and R. K. Snook. Basic Biology Course, Book 2: Electron Microscopy and Cell Structure. Cambridge, 1975.
Electron Microscopy of Enzymes: Principles and Methods, vols. 1–2. New York, 1973–74.

N. A. STAROSVETSKAIA and IA. IU. KOMISSARCHIK