The principle of Transmission Electron Microscope (TEM)

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To satisfy this curiosity, many inventions have been devised. One of them is the optical microscope. The human eye can distinguish objects down to about 0.2 mm. Optical microscopes reveal small objects, which would be otherwise invisible to the human eye, by magnifying them with the help of a combination of glass lenses. If we raise the amplification rate (magnification) of an optical microscope higher and higher, can we see an atom?

Unfortunately, the answer is “NO.” Optical microscopes use light as the illumination, so they have a limited ability to distinguish small structures (resolution). They cannot distinguish any structure smaller than the wavelength of light.Engineers, like Ruska in Germany, broke this limit. They invented the “electron microscope”, which uses an electron beam as the illumination source instead of light. That enables us to observe small structures at a far better magnification than is possible with optical microscopes. It is now possible to distinguish the arrangement of atoms in materials.

Electron microscopes enable clear observation of micro-structures, which is not possible with optical microscopes. Moreover, they also make it possible to analyze substance structures and obtain atomic level information by using an electron beam. The electron microscope is an epoch-making invention used throughout the world to investigate an atomic world that we could hardly imagine.

The difference between Electron Beam and Light

A characteristic of electrons is that they cannot move freely in the air. They can, however, move freely in a vacuum. For this reason, a vacuum is maintained inside the column of an electron microscope; something that is not required for an optical microscope.A specimen is illuminated by a beam of electrons accelerated by a device called an electron gun. These electrons either penetrate the specimen or cause scattering. By selectively converging and diverging these electrons with an electron lens (electric and magnetic fields deflect the electron beam to form images, in the same way the glass lenses deflect the light for the optical microscope), the enlarged images are formed on a fluorescent surface which is positioned below the beam and specimen.Electron beams are flows of electrons generated in the vacuum by heating or by applying a strong electric field to a fine filament, and have the nature of a “wave”, with a wavelength shorter than that of visible light. Instead of glass, the lenses of an electron microscope are a combination of electromagnets constructed to form magnetic field lenses.

fig1. Ripples caused by the difference in the magnitude of the wave

As explained above, the ability to distinguish a small structure, that is resolution, largely depends on the wavelength of the “wave” used to illuminate the specimen.

The nature of this “wave” may be easily understood by comparing it to the wave pattern arising when a small stone is thrown into a lake. Assume the waves on the water surface come into contact with a rock protruding above the surface. If the rock is larger than the length between the crests of the waves (wavelength), then the wave pattern does not continue behind the rock (Fig,1). This creates a shadow. If the rock is smaller than the wavelength, however, the wave pattern will not be interrupted behind the rock and there is no shadow. In this case, the existence of the rock cannot be detected.

Whereas the wavelength of visible light is 400 to 800 nm (1 nanometer is one 100,000th of 0.1mm), the wavelength of the electron beam, which is used as a light source in the electron microscope, varies depending on the accelerating voltage. The accelerating voltages commonly used are 100 to 200 kV (corresponding to wavelengths of 0.0037nm to 0.0025nm).

This wavelength is far shorter than that of light, and sufficient to distinguish the arrangements of atoms (several nanometers). For the optical microscope the combination of the lens is varied to alter the magnification. In contrast, for the electron microscope, the intensity of the electric current passed to the electromagnets is varied to change the intensity of the magnetic field. This corresponds to the changing the thickness of a convex lens. In fact, by manipulating the electric current, the magnification can be freely controlled.

Another characteristic “electron diffraction”

Another great feature of the electron microscope is that an electron diffraction pattern can be obtained.This is important information which reveals the nature of materials (specimen), especially, its atomic arrangement. Similar information can be obtained using an X-ray, but it lacks correlation with the image of the irradiated area. Electron microscopes allow images to be observed at a high magnification and diffraction analysis at a nanometer scale to be performed for the same irradiated area.Electrons used to illuminate a very thin specimen, will be scattered while penetrating it. This process gives an electron diffraction pattern and the electron diffraction method can reveal the arrangement of molecules and atoms in a crystalline specimen. This technique is playing an important role in the field of material science.

TEMs employ a high voltage electron beam in order to create an image. An electron gun at the top of a TEM emits electrons that travel through the microscope’s vacuum tube. Rather than having a glass lens focusing the light (as in the case of light microscopes), the TEM employs an electromagnetic lens which focuses the electrons into a very fine beam. This beam then passes through the specimen, which is very thin, and the electrons either scatter or hit a fluorescent screen at the bottom of the microscope. An image of the specimen with its assorted parts shown in different shades according to its density appears on the screen. This image can be then studied directly within the TEM or photographed.  Figure 1 shows a diagram of a TEM and its basic parts. 

Fig. 1 Simplified diagram of a transmission electron microscope.  Drawing by Graham Colm, courtesy of Wikimedia Commons.

What Are the Differences Between a TEM and a Light Microscope?

Although TEMs and light microscopes operate on the same basic principles, there are several differences between the two. The main difference is that TEMs use electrons rather than light in order to magnify images. The power of the light microscope is limited by the wavelength of light and can magnify something up to 2,000 times. Electron microscopes, on the other hand, can produce much more highly magnified images because the beam of electrons has a smaller wavelength which creates images of higher resolution. (Resolution is the degree of sharpness of an image.) Figure 2 compares the magnification of a light microscope to that of a TEM. 

Fig. 2 [left] Cotton stem; area in the circle is the phloem tissue. Light microscope x250. Photo by K. Esau.  [right] Enlarged image of cotton phloem tissue showing a sieve element (top cell) and a companion cell (bottom cell), TEM x8,000. Photo by J. Thorsch.

How Are TEM Specimens Prepared?

Specimens must be very thin so that electrons are able to pass through the tissue. This may be done by cutting very thin slices of a specimen’s tissue using an ultramicrotome.  The tissue must first be put in a chemical solution to preserve the cell structure.  The tissue must also be completely dehydrated (all water removed). 

                                 Fig. 3 Ultramicrotome.  Photo by J. Thorsch.                                                             Fig. 4 Microtome grid.  Image by Laurie Hannah

Once preserved and dehydrated, tissue samples are placed in hard, clean plastic.  The plastic supports the tissue while it is being thinly cut with the ultramicrotome (Fig. 3).

After sections are cut and mounted on grids, (tiny circular disks with openings,) a solution of lead is used to stain the tissue (Fig. 4).  The lead provides contrast to the tissue by staining certain cell parts.  When placed in the electron microscope, the electrons are scattered by the lead.  They do not penetrate the tissue or hit the fluorescent screen, leaving those areas dark. 

Esau’s Work With the TEM

Esau started using the TEM in her research in the early 1960s.  When she moved to UC Santa Barbara in 1963, the campus purchased a Siemens electron microscope for her. She then received a grant from the National Science Foundation in 1969 for another new microscope which she used for the remainder of her career in Santa Barbara. The TEM significantly improved her understanding of the relationship between plants and viruses. Electron microscopy also aided in clarifying the functioning of sieve elements, the food conducting cells in plants. Without the TEM, much of this research would not have been possible.