From the equation shown , you can see that if it were possible to use a shorter wave-length of light, we could improve the resolving power of a microscope. However, because we are limited by the wavelength of light visible to the human eye, we are not able to do this with the light microscope. The electron microscope is able to achieve greater magnification and resolution because it uses a high voltage beam of electrons, whose wavelength is very much shorter than that of visible light. Consequently we are able to resolve points that are much closer together than is possible even with the very best light microscope. The resolving power of an electron microscope may be as low as 1–2 nm, enabling us to see viruses, for example, and the internal structure of cells. The greatly im-proved resolution means that specimens can be meaningfully magnified over 100 000×.
where λ is the wavelength of the light source, n is the refractive index of the air or liquid between the objec-tive lens and the specimen and θ is the aperture angle (a measure of the light-gathering ability of the lens).
The expression n sinθ is called the numerical aperture and for high quality lenses has a value of around 1.4. The lowest wavelength of light visible to the human eye is approximately 400 nm, so the maximum resolving power for a light microscope is approximately
Electron microscopes, which were first developed in the 1930s and 1940s, use ring-shaped electromagnets as ‘lenses’ to focus the beam of electrons onto the specimen. Because the electrons would collide with, and be deflected by, molecules in the air, electron microscopes require a pump to maintain a vacuum in the column of the instru-ment. There are two principal types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM).
Figure 1.7 shows the main features of a TEM. As the name suggests, the electron beam passes through the specimen and is scattered according to the density of the different parts. Due to the limited penetrating power of the electrons, extremely thin sections (<100 nm, or less than one-tenth of the diameter of a bacterial cell) must be cut, using a diamond knife. To allow this, the specimen must be fixed and dehydrated, a process that can introduce shrinkage and distortion to its structure if not correctly performed.
After being magnified by an objective ‘lens’, an image of the specimen is projected onto a fluorescent screen or photographic plate. More dense areas, which scatter the beam, appear dark, and those where it has passed through are light. It is often necessary to enhance contrast artificially, by means of ‘staining’ techniques that involve coating the specimen with a thin layer of a compound containing a heavy metal, such as osmium or palladium. It will be evident from the foregoing description of sample preparation and use of a vacuum that electron microscopy cannot be used to study living specimens.
The TEM has been invaluable in advancing our knowledge of the fine structure of cells, microbial or otherwise. The resulting image is, however, a flat, two-dimensional one, and of limited use if we wish to learn about the surface of a cell or a virus. For this, we turn to SEM. The scanning electron microscope was developed in the 1960s and provides vivid, sometimes startling, three-dimensional images of surface structure. Samples are dehydrated and coated with gold to give a layer a few nanometres thick. A fine beam of electrons probes back and forth across the surface of the specimen and causes secondary electrons to be given off. The number of these, and the angle at which they are emitted, depends on the topography of the specimen’s surface. SEM does not have quite the resolving power of the TEM, and therefore does not operate at such high magnifications. Between them, SEM and TEM have opened up a whole new world to microbiologists, allowing us to put advances in our knowledge of microbial biochemistry and genetics into a structural context.