Transmission electron microscopy

Type of physical science: Condensed matter physics

Field of study: Surfaces

Transmission electron microscopes are magnifying instruments that use a beam of electrons as an illumination source. Electrons passing through the specimen are focused into a magnified image by magnetic lenses. The high resolution obtained by these instruments allows visualization of specimen details at molecular and even atomic levels.

Overview

A transmission electron microscope (TEM) is a magnifying instrument that uses a beam of electrons as a source of illumination for examining specimens. The word "transmission" refers to the fact that these microscopes form an image from electrons that pass through the specimen rather than being reflected from the surface or emitted. The electron beams used for illumination have relatively short wavelengths, allowing objects as small as individual atoms to be visualized. This is in contrast to light microscopes, which use the comparatively long wavelengths of visible or ultraviolet light as an illumination source; such microscopes are only capable of visualizing objects hundreds of times larger than those viewable with a transmission electron microscope.

TEMs resemble light microscopes in overall design. The primary differences between the two are the illumination source and the types of lenses used. A light microscope uses an incandescent light or the sun as an illumination source; transmission electron microscopes use an electron "gun" that emits a beam of electrons at a very short wavelength. In a light microscope, beams of light are focused by glass lenses; in transmission electron microscopes, the electron beam is focused by magnetic lenses. The magnetic fields of the lenses are generated by massive coils of wire through which a precisely controlled electric current is passed. This shapes the magnetic fields into the three-dimensional form necessary for focusing electrons. Although the pathways followed by electrons passing through the magnetic fields are complex, the net effect is the same as that of a glass lens on light rays: magnetic lenses focus the electron beam to a point, then expand the beam to project the resulting image for viewing.

The configuration of a typical TEM is inverted compared to that of a light microscope. The gun that emits electrons is at the top of the instrument, a series of magnetic lenses is in the middle, and the image is formed at the bottom. The electron gun at the top of the microscope consists of two major parts, a filament and an anode. The filament is a thin wire, often made of tungsten, that is heated to a high temperature by an electric current. The heat drives electrons from the surface of the filament. The filament and its holder, which are electrically insulated from the rest of the column, are maintained at a high negative voltage. The anode, a metal plate placed a few centimeters below the filament, is grounded and is thus positive with respect to the filament. As a consequence, electrons from the filament are strongly attracted to the anode. As they traverse the distance from the filament to the anode, the electrons are accelerated to a velocity that depends on the voltage difference between the two locations—the greater the voltage difference, the greater the velocity. The wavelength of the electron beam, in turn, is inversely proportional to the velocity obtained—the higher the velocity, the shorter the wavelength. In TEMs with a difference of fifty thousand to one hundred thousand volts between the filament and anode, the velocity attained produces electron wavelengths between 0.054 and 0.037 angstrom.

The electron beam passes through a small hole in the anode and is focused by a series of magnetic lenses located below the gun. Just below the gun, a condenser lens focuses the electron beam into a very small, intense spot. The specimen is placed at the level of the focused spot. Electrons passing through the specimen are focused into an image by a series of three lenses: the condenser, objective, and projector lenses. Modern TEMs typically also have intermediate lenses. The condenser lens (or lenses) narrows the electron beam, and subsequent lenses all contribute to the magnification of the image.

Rather than focusing by moving the lenses, as is done in the light microscope, the TEM is focused by altering the current that is applied to the wire coils of the magnetic lenses—that is, by altering the magnifying power rather than the positions of the lenses. In more technical terms, changes in the current applied to the lens coil alter the focal length of a magnetic lens. Electrons entering the lens in a parallel beam converge at a point just beyond the lens, which is called the focal point. Increasing the current applied to a magnetic lens moves the focal point closer to the lens and thus decreases the focal length. As the focal length becomes shorter, the magnifying power of the lens increases. Reducing the current has the opposite effect. All the magnetic lenses in a TEM have wide ranges of focal length and magnification.

The final lens (or lenses), the projector, focuses the magnified image onto a phosphorescent screen at the bottom of the lens column. This screen is coated with a layer of crystals that respond to electron bombardment by emitting visible light, thus converting the electron image to a visual image. Because photographic emulsions can be directly exposed by electrons as well as light, a photograph of the electron image can be made by placing a photographic plate at the level of the screen. Alternatively, the image can be captured digitally with a charge-coupled device (CCD) camera.

The effect of the specimen on the electron beam and the basis for image formation differ significantly from the equivalent processes in a light microscope. In a light microscope, different parts of the specimen absorb or delay the passage of light rays differently. These differences produce variations in color and contrast in the image. In a TEM, atoms in the specimen deflect or scatter individual electrons from their paths in the beam. Positively charged atomic nuclei scatter electrons by attracting them from their paths; negatively charged electron clouds around the atomic nuclei scatter electrons by repelling them. Materials with a relatively high atomic number, such as lead, gold, or uranium, scatter electrons to the greatest degree.

Electrons scattered widely enough by subparts of the specimen collide with the sides of a brass fitting inside the lens and are eliminated from the beam. This creates "holes" in the beam that correspond to areas of the specimen that contain heavier atoms. After the beam is magnified by the objective, intermediate, and projector lenses and focused on the specimen screen, the areas that contain heavier atoms appear darker because fewer electrons strike the screen in these regions. Thus, the image of the specimen is represented on the screen as differences in contrast. Because passage through the specimen does not significantly alter the wavelengths of electrons, the image seen in the transmission electron microscope is monochrome; that is, no differences are produced that are equivalent to the color variations in light microscope images. Although any single color could be used, electron images are usually printed in black and white for practical reasons.

Applications

The ability of a microscope of any kind to visualize small objects is called the resolution or resolving power of the instrument. The resolving power of electron microscopes is equivalent to 0.61λ / 0.5α, where 0.61 is a factor that evaluates the conditions necessary for visualization by the human eye, λ is the wavelength of the electron beam, and α the angle of the cone of electrons entering the objective lens. For the very best resolution, this relationship requires that the wavelength of the electron beam be as short as possible and the angle of the cone of electrons, called the angle of illumination, be as large as possible.

The shortest wavelengths of visible light that can be used as an illumination source for light microscopes are near four hundred nanometers. Using this value as the basis for calculating the best possible resolution of light microscopes, which follows a relationship similar to that of electron microscopes, gives an ultimate resolving power of about 0.2 micrometer, or 2,000 angstroms. Although about a thousand times greater than the resolution of the unaided human eye, this value is nowhere near the resolving power of a typical TEM, which is approximately 0.2 nanometer, or 2 angstroms.

Electrons, even those traveling at relatively high velocities, are easily deflected by gas molecules or specimen atoms. For this reason, the entire pathway traveled by electrons inside an electron microscope must be kept at a near-perfect vacuum, and the specimen must be very thin.

The image on the fluorescent screen is viewed through ports that consist of thick leaded-glass plates. Interlocks allow specimens and photographic plates to be exchanged without disturbing the vacuum in the instrument. In order to protect the operator from x-rays generated when stray electrons strike metal surfaces in the microscope, heavy metal shielding is placed around the column housing the gun, magnetic lenses, and viewing area.

TEMs are provided with controls for adjusting the current to the magnetic lenses. These controls focus the condenser lens precisely on the specimen and allow the total magnification produced by the objective, intermediate, and projector lenses to be varied at will. Other controls are connected to ultrafine screws that allow the specimen to be held stationary or to be moved over distances as small as a few angstroms.

Specimens to be viewed in TEMs must be dry and nonvolatile in order to avoid disturbing the operating vacuum. In order to make specimens thin enough to transmit electrons, they must be dried to a thin film or cut into ultrathin slices. The maximum useful thickness of a specimen depends on both the voltage and the desired resolution; generally, at the highest voltage and lowest resolution, thickness should be no greater than one micrometer, or 10,000 angstroms. Ideal thicknesses are about 500 to 1,000 angstroms.

These operating limitations place severe requirements on specimens, particularly those examined in applications of the transmission electron microscope to biology and medicine. Living specimens are far too thick and volatile to be examined directly in the microscope. Instead, specimens are prepared by one of two major techniques.

In the first preparation technique, specimens are fixed, embedded in plastic, and cut into very thin sections. Fixation involves reaction with reagents, such as formaldehyde or glutaraldehyde, that introduce chemical cross-links between specimen molecules. The cross-links stabilize the molecules and hold them in position during the process of embedding, sectioning, and examining the sample. As part of the embedding procedure, water and other volatile materials in the specimen are replaced by the molecules of the plastic in liquid form. Hardening or polymerization of the plastic converts it and the specimen into a stable, nonvolatile form that can be cut into ultrathin slices. In the other technique, a solution of molecules or structures is spread in a thin layer over a plastic film. After it has dried, the material is ready to be placed in the microscope.

Biological specimens prepared by either method produce little contrast in the electron microscope because the primary atoms of living tissues—carbon, oxygen, hydrogen, and nitrogen—do not scatter electrons to any appreciable degree. In order to increase the contrast, biological specimens are usually stained with a solution of a heavy metal compound such as lead citrate, osmium tetroxide, or uranyl acetate. Atoms of the heavy metal are deposited in and around specimen details, greatly increasing electron scattering in these regions. This outlines specimen details with a dark contrast that makes them clearly visible in the electron image. Another way to enhance the contrast and surface details of isolated specimens is by shadowing, in which a thin layer of a heavy metal is deposited on specimen surfaces. Molecules or structures dried on a plastic film are coated under a vacuum by a heavy metal such as gold or platinum. The coating is produced by heating a small quantity of the metal to the boiling point on an electrical filament located to one side of the specimen. Atoms of evaporated metal are deposited on the raised surfaces of the specimen facing the filament. These coated surfaces then produce high contrast in the resulting image.

The above methods, now applied routinely, produce specimens that reveal details at the molecular and even the atomic level. However, even greater resolution could be achieved by electron ptychography. While the resolving power of TEMs has advanced significantly over the decades, a microscope's resolution is ultimately limited by its need for lenses, as even the smallest imperfections in a lens will blur an image at the highest magnification. Electron ptychography avoids this problem by bypassing lenses altogether; instead of relying on the microscope to produce the image, scientists analyze and reconstruct the scattering of the electrons and use that information to create a much more accurate image of the sample. The technique was pioneered at the University of Sheffield and announced in 2012 by project leader John Rodenburg, who claimed that with further study, electron ptychography should be able to produce an image with a resolution of one-tenth the diameter of an atom.

Context

Light microscopes capable of a maximum resolution of 0.2 micrometer were constructed during the latter half of the nineteenth century. As it became obvious that the resolution of light microscopes is fundamentally limited to this level, physicists identified forms of radiation with shorter wavelengths as possible sources of illumination. The greatest possibilities were offered by electron beams, which could readily be produced at wavelengths much shorter than those of visible light. Intensive research into the problems of designing lenses capable of focusing electrons began in Germany during the late 1920s, leading to the development of the first practical transmission electron microscope by Ernst Ruska and Bodo von Borries in the 1930s. Commercial production of their microscope began in Germany shortly before the onset of World War II. These developments were paralleled in Canada, where James Hillier and Albert Prebus designed the first high-resolution transmission electron microscope in North America in 1938. By the late 1950s, specimen-preparation methods were sufficiently advanced to allow effective use of transmission electron microscopes in physical, chemical, and biological research.

Applications of transmission electron microscopes have been particularly effective in metallurgy, the biological sciences, and medicine. From about 1958 to 1970, the resolving power of the TEM revealed details of biological structure that were unimagined by earlier investigators who were limited by light microscopy. The TEM pushed structural investigations to the molecular level and was instrumental in starting the molecular revolution in the biological sciences.

Principal terms

ELECTRON GUN: a device that uses a filament and an anode to emit a beam of electrons

FOCAL LENGTH: the distance between the center of a converging lens and the point at which parallel rays entering the lens are brought to a point of focus

MAGNETIC LENS: a massive wire coil through which a current is passed to generate a magnetic field capable of focusing an electron beam

RESOLUTION: a measure of the ability of a microscope to render small details in the image as separately distinguishable points

SHADOWING: a method of preparing specimens for microscopic examination in which the surfaces of the specimen are coated with a layer of evaporated metal

WAVELENGTH: the distance between corresponding points, such as peaks or troughs, in successive waves of radiation

Bibliography

Bradbury, Savile. The Evolution of the Microscope. New York: Pergamon, 1967. Print. This highly readable book presents a complete description of the theory and development of the light microscope and continues with a historical survey of events leading to the transmission electron microscope. Many illustrations of light and electron lenses, their action, and microscopes of both types from earliest models to contemporary instruments are included.

Brydson, Rik, ed. Aberration-Corrected Analytical Transmission Electron Microscopy. Hoboken: Wiley, 2011. Print.

Dehm, Gerhard, James M. Howe, and Josef Zweck, eds. In-Situ Electron Microscopy: Applications in Physics, Chemistry and Materials Science. Weinheim: Wiley, 2012. Print.

"Scientists Revolutionize Electron Microscope: New Method Could Create Highest Resolution Images Ever." ScienceDaily. ScienceDaily, 6 Mar. 2012. Web. 23 Dec. 2013.

Sjöstrand, Fritiof S. Instrumentation and Techniques. Vol. 1 of Electron Microscopy of Cells and Tissues. New York: Academic, 1967. Print. Written by an individual who was involved in the development of instrumentation and preparative techniques in electron microscopy, this book describes the theory of the instrument and its applications in the biological sciences in detail. Although written at a technical level, it can easily be understood by readers who do not have a background in physics or mathematics.

Spence, John C. H. High-Resolution Electron Microscopy. 4th ed. Oxford: Oxford UP, 2013. Print.

Watt, I. M. The Principles and Practice of Electron Microscopy. New York: Cambridge UP, 1985. Print. A readable work that presents the theoretical background of electron microscopy in clear and understandable terms and outlines its applications, including methods used to prepare specimens for examination.

Williams, David B., and C. Barry Carter. Transmission Electron Microscopy: A Textbook for Materials Science. 2nd ed. 4 vols. New York: Springer, 2009. Print.

Wilson, Michael B. The Science and Art of Basic Microscopy. Bellaire: Amer. Soc. for Medical Technology, 1976. Print. Although concerned with light microscopes, this short book begins with basic optical principles and then deals with lens theory and the construction, operation, and applications of various types of microscopes. Clearly and simply written, it is easily understood by a nontechnical reader.

Wischnitzer, Saul. Introduction to Electron Microscopy. 3rd ed. New York: Pergamon, 1981. Print. This book explains the theory and operation of electron microscopes and gives step-by-step details of the major specimen-preparation techniques. This is a practical, understandable, and readable book that has served as a valuable introduction to electron microscopy for laypeople as well as scientists.

Charges and Currents

X-Ray and Electron Diffraction