Microscopes used for research commonly have a number of refinements to enable a complete study of the specimens. Because the image of a specimen is highly magnified and inverted, manipulating the specimen by hand is very difficult. As a result, the stages of high-powered research microscopes are mounted so that they can be moved by means of micrometer screws; in some microscopes, the stage can also be rotated. In addition, all research microscopes are equipped with three or more objectives, mounted on a revolving head, so that the magnifying power of the microscope can be varied.
III SPECIAL-PURPOSE OPTICAL MICROSCOPES
A number of types of microscopes have been developed for specialized uses. One such type is the stereoscopic microscope, which is actually two low-powered microscopes arranged so that they converge on the specimen. These instruments provide a three-dimensional image that has its right side up.
The ultraviolet microscope uses the ultraviolet region of the spectrum rather than the visual region, either to gain resolution because of the shorter wavelength or to emphasize detail by selective absorption at different wavelengths within the ultraviolet band. Because glass does not transmit the shorter ultraviolet wavelengths, the optics used in this type of microscope are usually quartz, fluorite, or aluminized-mirror systems. Further, because ultraviolet radiation is invisible, the image is made visible through phosphorescence (see Luminescence), photography, or electronic scanning. The ultraviolet microscope is used in medical research.
The petrographic microscope is used to identify and quantitatively estimate the mineral components of igneous rock and metamorphic rock. It is equipped with a Nicol prism or other polarizing device to polarize the light that passes through the specimen being examined (see Optics: Polarization of Light). Another Nicol prism or analyser determines the polarization of the light after it has passed through the specimen. The microscope also has a rotating stage that, by suitable adjustment, indicates the change in polarization caused by the specimen.
The dark-field microscope employs illumination in the form of a hollow, extremely intense cone of light, which is concentrated on the specimen. The field of view of the objective lies in the hollow, dark portion of the cone and thus picks up only scattered light from the object. As a consequence, the clear portions of the specimen appear as a dark background, and the minute objects under study glow brightly against this dark field. This form of illumination is useful for transparent, unstained biological material and for minute objects that cannot be seen in normal illumination under the microscope.
The phase microscope illuminates the specimen with a hollow cone of light, as in the dark-field microscope. In the phase microscope, however, the cone of light is narrower and enters the field of view of the objective. Within the objective is a ring-shaped device that both reduces the intensity of the light and introduces a phase shift of a quarter of a wavelength. This form of illumination causes minute variations of refractive index in a transparent specimen to become visible. This type of microscope is particularly effective for studying living tissue; hence, it is used widely in biology and medicine.
Very advanced optical microscopes include the near-field microscope, through which even details slightly smaller than the wavelengths of light can be seen. A light beam shining through a tiny hole is played across the specimen at a distance of only about half the diameter of the hole, until an entire image is obtained.
Electron
The magnifying power of an optical microscope is limited by the wavelength of visible light. An electron microscope uses electrons to “illuminate” an object; since electrons have a much smaller wavelength than light, they can resolve much smaller structures than light can. The smallest wavelength of visible light is about 4,000 angstroms (1 angstrom is .0000000001 metres); the wavelength of electrons used in electron microscopes is usually about .5 angstrom.
All electron microscopes comprise several basic elements. They have an electron gun emitting electrons that strike the specimen and create a magnified image. Magnetic “lenses” that create magnetic fields are used to direct and focus the electrons, because the conventional lenses used in optical microscopes to focus visible light do not work with electrons. A vacuum system is an important part of any electron microscope. Electrons are easily scattered by air molecules, so the interior of an electron microscope must be at a very high vacuum. Finally, electron microscopes also have a system that records or displays the image produced by the electrons.
There are two basic types of electron microscopes: the transmission electron microscope (TEM), and the scanning electron microscope (SEM). In a TEM, the electron beam is directed on to the object to be magnified. Some of the electrons are absorbed or bounce off the specimen; others pass through and form a magnified image of the specimen. The sample must be cut very thin to be used in a TEM; usually the sample is no more than a few thousand angstroms thick. A photographic plate or fluorescent screen is placed beyond the sample to record the magnified image. Transmission electron microscopes are capable of magnifying an object up to 1 million times.
A scanning electron microscope creates a magnified image of the surface of an object. When using an SEM, the object to be magnified does not need to be thinly sliced; the sample can be placed in the microscope with little, if any, preparation. An SEM scans the surface of the sample bit by bit, in contrast to the TEM, which looks at a relatively large part of the object all at once. In an SEM, a tightly focused electron beam moves over the entire sample, much the way an electron beam scans an image on to the screen of a television. Electrons in the tightly focused beam might scatter directly off the sample, or cause secondary electrons to be emitted from the surface of the sample; these scattered or secondary electrons are collected and counted by an electronic device located to the side of the sample. Each scanned point on the sample corresponds to a pixel on a television monitor; the more electrons the counting device detects, the brighter the pixel on the monitor is. As the electron beam scans over the entire sample, a complete image of the sample is displayed on the monitor. Scanning electron microscopes can magnify objects 100,000 times or more. SEMs are particularly useful because, unlike TEMs and powerful optical microscopes, SEMs produce detailed pictures of the surface of objects, providing a realistic three-dimensional image.
Various other electron microscopes have been developed. A scanning transmission electron microscope (STEM) combines elements of an SEM and a TEM, and can resolve single atoms in a sample. An electron probe microanalyser, which is an electron microscope fitted with an X-ray spectrum analyser, can examine the high-energy X-rays that are emitted by the sample when it is bombarded with electrons. Because the identity of different atoms or molecules can be determined by examining their X-ray emissions, electron probe analysers not only provide a magnified image of the sample as a conventional electron microscope does, but also information about the sample's chemical composition.
