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Pathology => Histology => Microscope
Microscope
INTRODUCTION
Microscope, instrument used to obtain a magnified image of minute objects or minute details of objects.
OPTICAL MICROSCOPES
The most widely used microscopes are optical microscopes, which use visible light to create a magnified image of an object. The simplest optical microscope is the double-convex lens with a short focal length (see Optics). Double-convex lenses can magnify an object up to 15 times.
The compound microscope uses two lenses, an objective lens and an ocular lens, mounted at opposite ends of a closed tube, to provide greater magnification than is possible with a single lens. The objective lens is composed of several lens elements that form an enlarged real image of the object being examined. The real image formed by the objective lens lies at the focal point of the ocular lens. Thus, the observer looking through the ocular lens sees an enlarged virtual image of the real image. The total magnification of a compound microscope is determined by the focal lengths of the two lens systems and can be more than 2000 times.
Optical microscopes have a firm stand with a flat stage to hold the material examined and some means for moving the microscope tube toward and away from the specimen to bring it into focus. Ordinarily, specimens are transparent and are mounted on slides-thin, rectangular pieces of clear glass that are placed on the stage for viewing. The stage has a small hole through which light can pass from a light source mounted underneath the stage-either a mirror that reflects natural light or a special electric light that directs light through the specimen.
In photomicrography, the process of taking photographs through a microscope, a camera is mounted directly above the microscope's eyepiece. Normally the camera does not contain a lens because the microscope itself acts as the lens system.
Microscopes used for research 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 difficult. Therefore, the stages of high-powered research microscopes can by moved by micrometer screws, and in some microscopes, the stage can also be rotated. Research microscopes are also equipped with three or more objective lenses, mounted on a revolving head, so that the magnifying power of the microscope can be varied.
SPECIAL-PURPOSE OPTICAL MICROSCOPES
Different microscopes have been developed for specialized uses. The stereoscopic microscope, two low-powered microscopes arranged to converge on a single specimen, provides a three-dimensional image.
The petrographic microscope is used to analyze igneous and metamorphic rock. A Nicol prism or other polarizing device polarizes the light that passes through the specimen. Another Nicol prism or analyzer determines the polarization of the light after it has passed through the specimen. Rotating the stage causes changes in the polarization of light that can be measured and used to identify and estimate the mineral components of the rock.
The dark-field microscope employs a hollow, extremely intense cone of light concentrated on the specimen. The field of view of the objective lens lies in the hollow, dark portion of the cone and picks up only scattered light from the object. The clear portions of the specimen appear as a dark background, and the minute objects under study glow brightly against the 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 also illuminates the specimen with a hollow cone of light. However, the cone of light is narrower and enters the field of view of the objective lens. Within the objective lens is a ring-shaped device that reduces the intensity of the light and introduces a phase shift of a quarter of a wavelength. This 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.
A typical optical microscope cannot resolve images smaller than the wavelength of light used to illuminate the specimen. An ultraviolet microscope uses the shorter wavelengths of the ultraviolet region of the light spectrum to increase resolution or to emphasize details by selective absorption. Glass does not transmit the shorter wavelengths of ultraviolet light, so the optics in an ultraviolet microscope are usually quartz, fluorite, or aluminized-mirror systems. Ultraviolet radiation is invisible to human eyes, so the image must be made visible through phosphorescence , photography, or electronic scanning.
The near-field microscope is an advanced optical microscope that is able to resolve details slightly smaller than the wavelength of visible light. This high resolution is achieved by passing a light beam through a tiny hole at a distance from the specimen of only about half the diameter of the hole. The light is played across the specimen until an entire image is obtained.
The magnifying power of a typical optical microscope is limited by the wavelengths of visible light. Details cannot be resolved that are smaller than these wavelengths. To overcome this limitation, the scanning interferometric apertureless microscope (SIAM) was developed. SIAM uses a silicon probe with a tip one nanometer (1 billionth of a meter) wide. This probe vibrates 200,000 times a second and scatters a portion of the light passing through an observed sample. The scattered light is then recombined with the unscattered light to produce an interference pattern that reveals minute details of the sample. The SIAM can currently resolve images 6500 times smaller than conventional light microscopes.
ELECTRON MICROSCOPES
An electron microscope uses electrons to "illuminate" an object. Electrons have a much smaller wavelength than light, so they can resolve much smaller structures. The smallest wavelength of visible light is about 4000 angstroms (40 millionths of a meter). The wavelength of electrons used in electron microscopes is usually about half an angstrom (50 trillionths of a meter).
Electron microscopes have an electron gun that emits electrons, which then strike the specimen. Conventional lenses used in optical microscopes to focus visible light do not work with electrons; instead, magnetic fields are used to create "lenses" that direct and focus the electrons. Since electrons are easily scattered by air molecules, the interior of an electron microscope must be sealed at a very high vacuum. Electron microscopes also have systems that record or display the images produced by the electrons.
There are two types of electron microscopes: the transmission electron microscope (TEM), and the scanning electron microscope (SEM). In a TEM, the electron beam is directed onto the object to be magnified. Some of the electrons are absorbed or bounce off the specimen, while 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 no more than a few thousand angstroms thick. A photographic plate or fluorescent screen beyond the sample records the magnified image. Transmission electron microscopes can magnify an object up to one million times.
In a scanning electron microscope, a tightly focused electron beam moves over the entire sample to create a magnified image of the surface of the object in much the same way an electron beam scans an image onto 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. 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.
An SEM scans the surface of the sample bit by bit, in contrast to a TEM, which looks at a relatively large area of the sample all at once. Samples scanned by an SEM do not need to be thinly sliced, as do TEM specimens, but they must be dehydrated to prevent the secondary electrons emitted from the specimen from being scattered by water molecules in the sample.
Scanning electron microscopes can magnify objects 100,000 times or more. SEMs are particularly useful because, unlike TEMs and powerful optical microscopes, they can produce detailed three-dimensional images of the surface of objects.
The scanning transmission electron microscope (STEM) combines elements of an SEM and a TEM and can resolve single atoms in a sample.
The electron probe microanalyzer, an electron microscope fitted with an X-ray spectrum analyzer, can examine the high-energy X rays emitted by the sample when it is bombarded with electrons. The identity of different atoms or molecules can be determined from their X-ray emissions, so the electron probe analyzer not only provides a magnified image of the sample, but also information about the sample's chemical composition.
SCANNING PROBE MICROSCOPES
A scanning probe microscope uses a probe to scan the surface of a sample and provides a three-dimensional image of atoms or molecules on the surface of the object. The probe is an extremely sharp metal point that can be as narrow as a single atom at the tip.
An important type of scanning probe microscope is the scanning tunneling microscope (STM). Invented in 1981, the STM uses a quantum physics phenomenon called tunneling to provide detailed images of substances that can conduct electricity. The probe is brought to within a few angstroms of the surface of the material being viewed, and a small voltage is applied between the surface and the probe. Because the probe is so close to the surface, electrons leak, or tunnel, across the gap between the probe and surface, generating a current. The strength of the tunneling current depends on the distance between the surface and the probe. If the probe moves closer to the surface, the tunneling current increases, and if the probe moves away from the surface, the tunneling current decreases. As the scanning mechanism moves along the surface of the substance, the mechanism constantly adjusts the height of the probe to keep the tunneling current constant. By tracking these minute adjustments with many scans back and forth along the surface, a computer can create a three-dimensional representation of the surface.
Another type of scanning probe microscope is the atomic force microscope (AFM). The AFM does not use a tunneling current, so the sample does not need to conduct electricity. As the metal probe in an AFM moves along the surface of a sample, the electrons in the probe are repelled by the electrons of the atoms in the sample and the AFM adjusts the height of the probe to keep the force on it constant. A sensing mechanism records the up-and-down movements of the probe and feeds the data into a computer, which creates a three-dimensional image of the surface of the sample.
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