microscope

an optical instrument used to produce greatly magnified images of objects (or parts of objects) that are invisible to the naked eye.

The human eye is a natural optical system characterized by a certain resolution—that is, the minimal distance between which elements of an observed object (perceived as points or lines) can still be distinguished from each other. For the normal eye, the minimal resolution at the minimal distance of distinct vision (D— 250 mm) is approximately 0.08 mm (in many cases, about 0.20 mm). Microorganisms, most plant and animal cells, small crystals, and the microstructural elements of metals and alloys are much smaller. Various types of microscopes are designed for the observation and study of such objects. The shape, size, structure, and many other characteristics of microscopic objects are determined by microscope. The microscope makes it possible to distinguish structures with only 0.20 micron (μ) between elements.

Historical survey. In the Netherlands and northern Italy as early as the 16th century, craftsmen making eyeglasses knew that a system of two lenses could be used to produce magnified images of objects. Some sources claim that a microscope-like instrument was constructed by Z. Janssen in the Netherlands in about 1590.

The rapid spread of microscopes and their improvement (chiefly by optical craftsmen) began in 1609–10, when Galileo, in the course of studying the optic tube he had designed, used it as a microscope by changing the distance between the objective and the eyepiece.

The first brilliant advances in the application of the microscope to scientific research are associated with the names of R. Hooke, who established the cellular structure of animal and plant tissues (c. 1665), and especially A. van Leeuwenhoek, who discovered microorganisms (1673–77). Microscopes first appeared in Russia in the early 18th century, when L. Euler (1762; Dioptrica, 1770–71) devised methods of computing their optical assemblies. G. B. Amici first used an immersion objective in a microscope in 1827. In 1850, the English optician H. Sorby built the first microscope for observing specimens in polarized light.

The scientific work of E. Abbe, who in 1872–73 developed the now classic theory of image formation in the microscope with non-self-luminous objects, contributed greatly to the extensive development of methods of microscopy and to the improvement of various types of microscopes. The Austrian researchers R. Zsigmondy and H. Siedentopf developed the ultramicroscope in 1903. In 1935, F. Zernicke proposed the phase-contrast method for observing through the microscope transparent objects that scatter light poorly. Soviet scientists, including L. I. Mandel’shtam, D. S. Rozhdestvenskii, A. A. Lebedev, and V. P. Linnik, have made a major contribution to the theory and practice of microscopy.

Optical system operating principles, magnification, and resolving power. A diagram of a typical microscope is presented in Figure 1. The specimen to be examined (7) is placed on a slide (10). A condenser (6) concentrates a beam of light reflected from a mirror (4) onto the specimen. A special illuminator, consisting of a lamp (1) and a collecting lens (2), generally serves as the light source. (A mirror sometimes directs ordinary daylight onto the specimen.) A field diaphragm (3) and an iris diaphragm (5) restrict the light beam and decrease the amount of extraneous scattered light striking the specimen.

Figure 1. Diagram of typical microscope

The appearance of the image of a specimen in a microscope can be described in basic (although extremely simplified) terms within the framework of geometric optics. Light beams originating at the specimen (7) are refracted in the objective (8) and create an inverted and magnified real optical image (7’) of the specimen. The image is viewed through an eyepiece (9). The microscope is focused so that the real optical image is located directly beyond the front focus of the eyepiece (Foe). Under these conditions, the eyepiece functions as a magnifying glass: in producing additional magnification, it forms a virtual image (7”; inverted, as before). Upon passing through the optical medium of the observer’s eye, the beams from the virtual image create a real image of the specimen on the retina. The virtual image is usually located at the minimal distance of distinct vision D from the eye. If the eyepiece is moved in such a way that the real image is in front of F_oc, then the image produced by the eyepiece becomes real and can be produced on a screen or photographic film. Microscopic objects are photographed and filmed in this manner.

The total magnification of a microscope is equal to the product of the linear magnification of the objective and the angular magnification of the eyepiece: = Δ._OC. The objective magnification Δ = /f_ob, where δ is the distance between the rear focus of the objective F’_ob and the front focus of the eyepiece (the optical length of the body tube of the microscope) and f’_ob is the focal length of the objective. The eyepiece magnification, like that of a magnifying glass, is expressed by the formula _oc = 250/f_oc (f’_oc given in millimeters). Objectives usually have magnifications from 6.3 to 100; eyepieces, from 7 to 15 (the values engraved on the mounting). Therefore, the total magnification of the microscope ranges from 44 to 1,500.

Technically, of course, it is possible to use objectives and eyepieces that produce a total magnification much greater than 1,500. Usually, however, this is inexpedient. High magnification is not in itself a goal. The function of a microscope is to make it possible to distinguish the smallest possible structural elements of a specimen—that is, to make maximum use of the microscope’s resolving power. However, resolving power is limited by the wave properties of light. (Although these properties are ignored in geometric optics, within whose framework image production in a microscope has so far been discussed, they are precisely what determines a microscope’s limitations.)

According to general principle, it is impossible when observing a specimen under illumination with a given wavelength X to distinguish elements of the specimen separated by distances much smaller than X. This principle is also manifested with the microscope, its quantitative expression differing slightly for self-luminous and non-self-luminous objects. Even when produced by an ideal (distortionless) objective, the image of a point emitting monochromatic light is not perceived by the eye as a point: because of light diffraction, it is actually a round spot of light of finite diameter d surrounded by several alternately dark and light rings. This image is called a diffraction spot, Airy spot, or Airy disk; d = 1.22λ/A, where λ is the wavelength. (When the specimen is illuminated in nonmonochromatic light, λ is usually the shortest wavelength of the light, or the wavelength at which the intensity of the irradiation is maximal.) In the same formula for the finite diameter d, A is the numerical aperture of the objective (A = n . sin u_m, where n is the refractive index of the medium separating the luminous point and the objective and um is half the aperture angle of the light beam emanating from the point and striking the objective).

When two luminous points are located close to each other, their diffraction patterns are superimposed, producing in the image plane a complex illumination distribution (see Figure 2). The least relative difference in illumination visible to the eye is 4 percent. This corresponds to the shortest distance between points at which the images of the points can be discerned (the maximum resolution of the microscope δ_max = 0.42d = 0.51 λ/A). As was shown in Abbe’s classic microscope theory, for non-self-luminous objects the maximum resolution δ_max = δ/(A + A’) where A and A’ are the numerical apertures of the objective and the condenser (the aperture values being indicated on the mounting).

Figure 2. Distribution of illumi-nation intensity in image of two proximate “points” in limiting case of visual resolution

The image of any specimen consists of the sum of the images of its structural elements. The smallest of these are perceived as points, the limitations resulting from light diffraction in micro-scopes being completely applicable here. When distances between points are less than the maximum resolution of the microscope, the points merge and cannot be seen separately. The resolving power of a microscope can be increased significantly only by increasing A. In turn, A can be increased only by increasing the refractive index n of the medium between the specimen and the objective (since sin u_m ≲ 1). This has been accomplished in immersion systems, whose numerical apertures reach A = 1.3 (in ordinary “dry” objectives, the maximum A ≈ 0.9).

The existence of a resolving power limit has an effect on the selection of magnifications. The range from 500 A to 1,000 A is considered effective magnification, since at these powers the observer’s eye can distinguish all of the structural elements of a specimen that are resolvable by the microscope. Beyond this point, the capabilities of the microscope with respect to resolving power are exhausted. No new structural details are revealed at magnifications above 1,000 A, although such magnifications are sometimes used in microphotography, image projection onto screens, and certain other cases. The electron microscope has a significantly greater resolving power and, consequently, a significantly higher effective magnification than has the ordinary microscope.

Methods of illumination and observation (microscopy). The structure of a specimen can be discerned only when its particles absorb or reflect light differently or differ in refractive index from each other or from the surrounding medium. These properties account for the amplitude and phase differences of light waves that have passed through different parts of a specimen (the determinant, then, of image contrast). Therefore, methods of observation with a microscope are designed and selected according to the character and properties of the specimens to be studied.

LIGHT FIELD IN TRANSMITTED LIGHT. The method of a light field in transmitted light is used in studies of transparent specimens with incorporated light-absorbing particles and components. Examples of such specimens include thin stained slices of animal and plant tissues and thin sections of minerals. In the absence of a specimen, the light beam from the condenser (6, in Figure 1) passes through the objective (8) to produce a uniformly illuminated field near the focal plane of the eyepiece (9). If an absorptive element is present in the specimen (7), it partially absorbs and partially scatters the incident light (broken line) and results in an image. This method can also be useful in observing nonabsorptive specimens, but only if they scatter the illuminating beam so strongly that a large part of it fails to strike the objective.

OBLIQUE ILLUMINATION. The method of oblique illumination is a variation of the light field in the transmitted light method, differing in that the light is directed onto the specimen at a large angle to the direction of observation. In many cases, this makes it possible to reveal the “relief of the specimen, a result of shadow formation.

LIGHT FIELD IN REFLECTED LIGHT. A light field in reflected light is used to observe nontransparent light-reflecting specimens, such as thin sections of metals or ores (see Figure 3). Illumination of the specimen (4) from an illuminator (1) and a semitransparent mirror (2) is from above, through the objective (3), which simultaneously functions as a condenser. The structure of the specimen can be seen in the image created in the image plane (6) by the objective and the tube lens (5) because of the difference in the reflecting power of its elements. Irregulari-ties that scatter the incident light are also revealed in a light field.

DARK FIELD IN TRANSMITTED LIGHT. A dark field in transmitted light is used to produce images of transparent nonabsorptive specimens that are invisible when illuminated by the light-field method (see Figure 4). Biological specimens are frequently of this type. Light from an illuminator (1) is directed by a mirror (2) onto the specimen by a specially designed dark-field condenser (3). Emerging from the condenser, most of the light rays, which have not changed direction in passing through the transparent specimen, form a beam in the shape of a hollow cone and fail to strike the objective (5; located within the cone). The image in the microscope is created by only a small portion of the

Figure 3. Light field in reflected light

rays: those that have been scattered by the microparticles of the specimen on the slide (4) into the cone and have passed through the objective. Light images of the structural elements of the specimen that differ from the surroundings in refractive index can be seen in the field of view (6) against a dark background. For large particles, only the light edges, which scatter the light rays, can be seen. The dark field in transmitted light cannot be used to determine whether particles are transparent or opaque or whether their refractive index is larger or smaller than that of the surroundings.

Figure 4. Dark field in transmitted light

ULTRAMICROSCOPY. The principle of the dark field in transmitted light is also the basis for the method of ultramicroscopy. Specimens in ultramicroscopes are illuminated perpendicularly to the direction of observation. The method makes it possible to detect (but not to see, in the literal sense) extremely small particles whose dimensions are far less than the maximum resolving power of the strongest microscopes. The presence of particles as small as 2 X 10^-9 m can be detected by means of immersion ultramicroscopes. It is impossible, however, to determine the shape and exact size of such particles by this method. The observer sees their images in the form of diffraction spots whose dimensions depend not on the size and shape of the particles themselves but on the aperture of the objective and the magnification of the microscope. Since such particles scatter very little light, extremely strong light sources (for example, carbon arcs) are required to illuminate them. Ultramicroscopes are used chiefly in colloid chemistry.

DARK FIELD IN REFLECTED LIGHT. A dark field in reflected light is used for opaque specimens, such as sections of metals. The specimen is illuminated from above, through a special annular system (an epicondenser) located around the objective.

POLARIZED LIGHT METHOD. Polarizing microscopy is used for the study of specimens containing or consisting entirely of optically anisotropic elements (many minerals, grains in sections of alloys, and certain animal and plant tissues). The optical properties of anisotropic objects are different in different directions and manifested in a variety of ways, depending on the orientation of the specimen with respect to the direction of observation and the plane of polarization of the incident light. The specimens can be observed in either transmitted or reflected light. The light projected by the illuminator passes through a polarizer. The polarization imparted to the light changes as the light passes through or is reflected from the specimen; these changes are studied with an analyzer and various optical compensators. From such changes, the basic optical characteristics of anisotropic microobjects can be assessed (the degree of birefringence, the number and orientation of optical axes, the rotation of the plane of polarization, and dichroism).

PHASE-CONTRAST METHOD. The phase-contrast method and a variation of it called anoptral contrast are used to obtain images of transparent and colorless specimens, such as unstained live animal tissues, which are invisible when observed by the light-field method. The basis for the method is that even when the difference in the refractive indexes of different elements of the specimen is very small, a light wave transmitted through these elements undergoes different phase changes (that is, acquires phase relief). These phase changes, which can be directly perceived neither by the eye nor on a photographic plate, are transformed by a special optical device into changes in the amplitude of the light wave—that is, into changes in brightness (the “amplitude relief), which are discernible to the eye and can be recorded on a photosensitive film. In other words, the brightness (amplitude) distribution in the resultant visible image duplicates the phase relief. Such an image is called a phase-contrast image.

In a typical diagram of this method (Figure 5), an iris diaphragm (2) with an annular opening is installed at the front focus of the condenser (3). The image of the iris diaphragm appears near the rear focus of the objective (5), where a phase plate (6) whose surface has an annular ridge or groove (a phase ring) is installed. The phase plate may or may not be placed in the focal plane of the objective (the phase ring often being made on the surface of one of the objective lenses). In either case, rays from the illuminator (1) that are undeflected in the specimen (4) and produce the image of the iris diaphragm (2) must pass completely through the phase ring, which (being absorptive) greatly attenuates them and changes their phase by A/4 (where X is the wavelength). At the same time, rays (even those that are only slightly deflected or scattered in the specimen) pass through the phase plate and bypass the phase ring (broken lines) without undergoing an additional phase shift. Taking into account the phase shift in the specimen, the total phase difference between deflected and undeflected rays proves to be near 0 or X/2; as a result of light interference in the image plane (4’) of the specimen (4), the rays noticeably amplify or attenuate each other and

Figure 5. Diagram of phase-contrast microscope

produce a contrast image of the structure of the specimen. The deflected rays have a considerably lower amplitude than do the undeflected rays; therefore, by making the amplitude values closer, the attenuation of the primary beam in the phase ring also leads to greater image contrast.

The phase-contrast method makes it possible to distinguish small structural elements that show exceedingly little contrast in the light-field method. Comparatively large transparent particles scatter light rays at such small angles that deflected rays pass through the phase ring together with undeflected rays. For such particles, there is a phase-contrast effect only near the contours, where significant scattering takes place.

INTERFERENCE-CONTRAST METHOD. In interference microscopy, every beam entering the microscope is bisected. One of the resultant beams is directed through the observed particle; the second bypasses the particle along the same or a supplementary optical path of the microscope. The beams are recombined in the eyepiece area and interfere with each other. The result of this interference is determined by the path difference of the beams 8, which is expressed by the formula δ = Nλ = (n₀—n_m)d, where n₀ and n_m are the refractive indexes of the particle and the surrounding medium, respectively; d is the particle thickness; TV is the order of interference; and λ is the wavelength.

A diagram of one of the methods of interference contrast is presented in Figure 6. The condehser (1) and the objective (4) are fitted with double-refracting plates (marked with diagonal arrows in the figure), the first splitting the original light beam into two; the second, recombining the beams. One of the beams, upon passing through the specimen (3), is phase delayed—that is, it acquires a path difference relative to the second beam. The extent of the delay is measured by the compensator (5).

Figure 6. Interference-contrast method

The interference-contrast method is similar in some respects to the phase-contrast method: both are based on the interference of beams that have passed through and bypassed the microparticle. Like the phase-contrast method, interference-contrast microscopy makes it possible to observe transparent and colorless objects, although the images may also be multicolored (interference colors). Both methods are suitable for the study of living tissues and cells and are frequently used for this purpose. The main distinguishing feature of the interference method is that, using compensators, it is possible to measure with high accuracy (to 1/300X) the path differences introduced by the microobjects. This presents broad opportunities for quantitative studies—for example, for calculation of the total mass and concentration of dry matter in a microscopic specimen, such as a plant or animal cell; of the refractive index; and of the size of the specimen (see Figure 7).

The interference-contrast method is frequently combined with other methods—in particular, with polarizing microscopy. Its use in conjunction with ultraviolet microscopy makes it possible to determine the nucleic acid content of the total dry mass of a specimen. Methods involving microinterferometers are also usually classified under interference microscopy.

Figure 7. Micrograph of human erythrocyte in monochromatic light (λ 0.546μ). Bend in interference band reproduces the thickness of the erythrocyte to scale

LUMINESCENCE METHOD. Luminescence, or fluorescence, microscopy consists in observing microscopic specimens under the green-orange luminescence produced when the specimens are illuminated with blue-violet or invisible ultraviolet light. There are two light filters in the optical diagram of such microscopes. The first is placed in front of the condenser and allows only those wavelengths to pass that stimulate the luminescence of either the specimen itself (natural luminescence) or of special dyes introduced into the specimen and absorbed by its particles (secondary luminescence). The second light filter, which is mounted behind the objective, transmits only the luminescent light to the observer’s eye or the photosensitive film. Specimens can be illuminated either from above, through the objective (which, in this case, also acts as a condenser), or from below, through an ordinary condenser. Illumination from above is sometimes called reflected light luminescence microscopy (an arbitrary term, since the excitation of the fluorescence of the specimen is not a simple reflection of the light). The method is frequently combined with phase-contrast observation in transmitted light.

Luminescence microscopy is used extensively in microbiology, virology, histology, cytology, the food industry, soil studies, microchemical analysis, and flaw detection. The abundance and diversity of its uses are associated with the extraordinarily high color sensitivity of the eye, the high image contrast of self-luminous specimens against dark, nonluminescent backgrounds, and the great value of the data that can be obtained concerning the composition and properties of substances, when the intensity and spectral composition of their luminescent radiation are known.

ULTRAVIOLET METHOD. Ultraviolet microscopy makes it possible to increase the maximum resolving power of a microscope—that is, to lower its minimal discernible value, which, as already mentioned, depends on the wavelength λ of the radiation used (for the ultraviolet rays used with a microscope, λ = 400–250 nanometers [nm]; for visible light, λ = 700–400 nm). This method expands the possibilities of microscopic research, since the particles of many substances that are transparent in visible light nevertheless strongly absorb ultraviolet radiation of certain wavelengths and are consequently easily distinguishable in ultraviolet images. A number of substances present in plant and animal cells (for example, purine and pyrimidine bases, most vitamins, aromatic amino acids, certain lipids, and thyroxine) have characteristic absorption spectra in the ultraviolet region. This accounts for the extensive use of ultraviolet microscopy in cytochemical analysis.

Ultraviolet rays are invisible to the human eye. Therefore, the images in ultraviolet microscopy are recorded either photographically or with an image converter or luminescent screen.

The following method for the color presentation of such images is widely used. The preparation is photographed at three wavelengths in the ultraviolet region of the spectrum. Each of the resultant negatives is illuminated with visible light of a particular color (blue, green, or red), and all three are projected simultaneously onto a single screen. A color image of the specimen in standard colors (depending on the absorptivity of the specimen in ultraviolet light) is created on the screen.

INFRARED METHOD. Like the ultraviolet method, infrared microscopy calls for the conversion of an invisible image into a visible one by photography or with an image converter. Infrared microscopy makes it possible to study the internal structure of specimens that are opaque in visible light, such as dark glass and certain crystals and minerals.

MICROPHOTOGRAPHY AND CINEMICROGRAPHY. The production with a microscope of images on light-sensitive films is used widely in conjunction with all other methods of microscopic research. In microphotography and cinemicrography, the optical system of the microscope requires some adjustment—an eyepiece focus, with respect to the objective image, that differs from the focus used for visual observation. Many modern microscopes have permanent (mounted) devices for microphotography that make it possible to make such adjustments and to project the images onto a photographic plate or film. Most microscopes can also be fitted with accessories for this purpose. Microphotography is indispensable for substantiating research, studying specimens in invisible ultraviolet and infrared rays, and studying specimens with weak luminescence intensity. Cinemicrography is important in studies of processes that occur over a period of time, such as the vital activities of tissue cells and microorganisms, the growth of crystals, and the course of simple chemical reactions.

Principal assemblies. In most microscopes (with the exception of the inverted type, to be discussed), a device for securing the objective is placed above, and a condenser below, the stage. All microscopes have a body tube in which the eyepieces are mounted. Coarse and fine focus mechanisms (changing the relative positions of specimen, objective, and eyepiece) are also essential parts of any microscope. All of these assemblies are attached to the microscope stand or frame.

The type of condenser used depends on the method of observation selected. Light-field condensers and phase-contrast and interference-contrast condensers are two- or three-lens systems that are very different from each other. Light-field condensers, whose numerical aperture may reach 1.4, contain an iris diaphragm that can be moved aside to allow oblique illumination of the specimen. Phase-contrast condensers are equipped with annular diaphragms. Dark-field condensers are complex systems of lenses and mirrors. Epicondensers—systems of annular lenses and mirrors mounted around the objective for dark-field observation in reflected light—constitute a special group. Special mirror-lens (catadioptric) and lens condensers that are transparent for ultraviolet rays are used in ultraviolet microscopy.

In most modern microscopes, the objectives are detachable and are selected according to the particular observation conditions. Often, there are several objectives in a single rotating (turret) head; the objective can be changed simply by turning this head.

A distinction is made between achromatic and apochromatic objectives, according to the degree of correction for chromatic aberration. Achromatic objectives are simpler in design. They correct for chromatic aberration in only two wavelengths, so that when the specimen is illuminated in white light the image remains slightly colored. Apochromatic lenses correct for three wavelengths and produce achromatic images. The image plane with achromatic and apochromatic lenses is somewhat curved. With visual observation, the accommodation of the eye and the possibility of seeing the entire field by readjusting the microscope compensate somewhat for this shortcoming. The phenomenon has a strong effect, however, in microphotography, in which the edges of the image are insufficiently sharp. Plane-achromats and plane-apochromats, objectives that make additional corrections for field curvature, are therefore widely used. Special projection systems that are inserted instead of the eyepiece to correct for curvature of the image surface are used in combination with ordinary objectives. These systems are unsuitable for visual observation.

Objectives are also classified according to (1) their spectral characteristics (those used for the visible region of the spectrum and those for ultraviolet and infrared microscopy [lens or mirror-lens objectives]); (2) the length of the body tube for which they are designed (those for a 160-mm tube; those for a 190-mm tube; and those for an “infinite length” tube, which create an image at infinity and are used together with an additional “body-tube” lens, which transfers the image to the focal plane of the eyepiece); (3) the medium between the objective and the specimen (dry and immersion types); (4) the method of observation (for example, normal, phase-contrast, or interference); and (5) the type of specimen (those for use with a cover slip or without). A special type is the epiobjective, a combination of a normal objective and an epicondenser.

The diversity of objectives has to do with the diversity of methods of microscopic observation, the variety of microscope design, and differences in the need for aberration correction under various operating conditions. Each objective is used only under those conditions for which it is designed. For example, an objective designed for a 160-mm body tube cannot be used in a microscope with a 190-mm body tube, and specimens without a cover slip cannot be observed with an objective designed to be used with a cover slip. It is especially important to adhere to the design conditions when working with dry objectives of large apertures (A > 0.6), which are very sensitive to any deviations from normal. Where cover slips are used, the cover-slip thickness should be 0.17 mm. An immersion objective may be used only with the immersion for which it is designed.

The type of eyepiece used with a given method of observation is governed by the choice of objective. A Huygens eyepiece is used with achromatic lenses of low and moderate magnification; a compensation eyepiece, which is designed so that its residual chromatic aberration is of a sign opposite that of the objective (to improve image quality), is used with apochromatic and achromatic lenses of high magnification. There are also special eyepieces used for photography and projection, which project the image onto a screen or photographic plate. Quartz eyepieces that are transparent for ultraviolet rays constitute a separate group.

The diverse accessories available for microscopes make it possible to improve the observation conditions and expand the possibilities for research. Illuminators of various types are designed to create optimal lighting conditions, ocular micrometers are used to measure the size of the specimens, and binocular tubes make it possible to observe a specimen with both eyes simultaneously. Photographic attachments and apparatus are used in microphotography, and drawing units make it possible to sketch the images. Special devices, such as microspectrophotometric attachments, are used for quantitative studies.

Types. The design of a microscope and its attachments and the characteristics of its main assemblies are determined either by the field for which the microscope is used, the range of problems, and the kind of specimens studied or by the method or methods of observation, or by both. Various types of specialized microscopes have therefore been developed to make it possible to study, with high precision, strictly defined classes of objects (or even certain of their properties). On the other hand, there are universal microscopes with which a variety of objects can be observed by a variety of methods.

BIOLOGICAL MICROSCOPES. Among the most widely used microscopes are those developed for botanical, histological, cytological, microbiological, and medical research and for observing transparent specimens in chemistry, physics, and other nonbio-logical fields. There are many models of biological microscopes, of differing design and accessories, which greatly expand the range of specimens that can be studied. The accessories include detachable illuminators providing transmitted and reflected light, detachable condensers for working with light and dark fields, phase-contrast devices, ocular micrometers, photographic attachments, and sets of light filters and polarizing devices that make it possible to use luminescence and polarization techniques with ordinary (nonspecialized) microscopes. The devices of microscopic technology used for readying specimens and conducting various operations on them (including work during observation) are especially important among the auxiliary equipment for biological microscopes.

Biological research microscopes are equipped with a set of interchangeable objectives, including epiobjectives for reflected light and, frequently, phase-contrast objectives, for various conditions and methods of observation and various types of specimens. There is a separate set of eyepieces (for visual observation and microphotography) suitable for each set of objectives. Biological research microscopes usually have binocular body tubes for observation with both eyes.

In addition to general-purpose microscopes, extensive use is also made in biology of various microscopes that are specialized according to the method of observation.

INVERTED MICROSCOPES. Inverted microscopes are distinctive in that the objective is located below the specimen, and the condenser, above. The path direction of the rays, transmitted from above downward through the objective, is altered by a system of mirrors such that they usually reach the eye from below (see Figure 8). Microscopes of this type are designed for the study of cumbersome specimens that are difficult or impossible to place on the stage of an ordinary microscope. Examples include tissue cultures in nutrient medium that are kept in a thermostatic chamber to maintain a preset temperature. Inverted microscopes are also used to study chemical reactions and to determine melting points. They are used when cumbersome auxiliary equipment is required for the observation of various processes. Inverted microscopes for microphotography and cinemicrography are fitted with special devices and cameras.

Figure 8. Diagram of the optical system of an inverted microscope

The inverted microscope is especially convenient for the observation of structures of various surfaces in reflected light. It is therefore used in most metallographic microscopes: the specimen (a section of a metal, alloy, or mineral) is mounted on the stage, polished surface downward; the nature of the rest of the sample is arbitrary and does not require treatment. Metallographic microscopes are also made in which the specimen is inserted from below, secured to a special plate. The assemblies in such microscopes are in the same relative positions as in ordinary (uninverted) microscopes. The surface under study is often etched beforehand, so that the grains of its structure become sharply distinguishable. Metallographic microscopes can be used with the light-field method, direct and oblique lighting, the dark-field method, and polarization. With work in a light field, the objective also acts as a condenser. Parabolic mirror epicondensers are used for dark-field illumination. A special attachment makes it possible to achieve phase contrast in a metallographic microscope with an ordinary objective.

LUMINESCENCE MICROSCOPES. Luminescence microscopes are equipped with a set of detachable light filters. By choosing the proper filter, it is possible to single out the part of the spectrum in the illuminator’s radiation that excites the luminescence of the specimen under study. A light filter can also be selected that only lets through luminescent light from the specimen. The luminescence of many objects is excited by ultraviolet radiation or the shortwave part of the visible spectum. Superhigh-pressure mercury vapor lamps produce precisely this type of radiation (and very brightly) and are therefore used as light sources. In addition to special luminescence microscopes, there are luminescence devices that are used in conjunction with ordinary microscopes. These contain an illuminator with a mercury vapor lamp, a set of light filters, and an opaque illuminator (for lighting specimens from above).

ULTRAVIOLET AND INFRARED MICROSCOPES. Ultraviolet and infrared microscopes are used for research in the invisible regions of the spectrum. Their optical diagrams are analogous to those of ordinary microscopes. Since making aberration corrections in the ultraviolet and infrared regions is very complicated, the condenser and objective of such microscopes are often catadioptric systems in which chromatic aberration is significantly reduced or eliminated altogether. The lenses are made of materials that are transparent to ultraviolet (quartz, fluorite) or infrared (silicon, germanium, fluorite, lithium fluoride). The microscopes are equipped with cameras in which the invisible images are fixed. Visual observation through the eyepiece in normal (visible) light is used where possible only for prefocusing and preadjusting the specimen in the microscope’s field of view. As a rule, ultraviolet and infrared microscopes have image converters to make invisible images visible.

POLARIZING MICROSCOPES. Polarizing microscopes are designed for the study (with optical compensators) of changes in the polarization of the light transmitted through or reflected from a specimen. This opens up opportunities for the quantitative and semiquantitative determination of various characteristics of optically active specimens. The assemblies of such microscopes are usually made so as to facilitate precise measurements: the eyepieces are equipped with cross hairs and a micrometric scale or reticule; the rotating stage has an angularly graduated dial for measuring the angle of rotation; and a Fedorov stage is often secured to the main stage, making it possible to rotate and tilt the specimen at will in order to find its crystallographic and crystallooptic axes. The objectives of polarizing microscopes are specially chosen for an absence of internal stresses, which lead to depolarization. Such microscopes usually contain a detachable auxiliary (Bertrand) lens to be used for observation in transmitted light. The Bertrand lens makes it possible to view the interference figures formed in the objective’s rear focal plane by the light, after it passes through the crystal under study.

INTERFERENCE MICROSCOPES. Transparent specimens are observed by the interference-contrast method with interference microscopes. Many interference microscopes are analogous in design to ordinary microscopes, differing only in the presence of a special condenser, objective, and measuring unit. If the specimens are observed in polarized light, the microscopes are equipped with a polarizer and an analyzer. Since they are used chiefly for biological research, interference microscopes may be classified as specialized biological microscopes. Microinterferometers (microscopes of a special type that are used to study the microrelief of surfaces of finished metal parts) are often classified as interference microscopes.

STEREOSCOPIC MICROSCOPES. Although they make possible the convenience of observation with both eyes, the binocular tubes used in ordinary microscopes do not produce a stereoscopic effect: the same rays enter both eyes at the same angles, only separated into two beams by a prism system. Stereoscopic microscopes, which make for a truly three-dimensional perception of the microscopic specimen, are actually two microscopes in a single unit, made in such a way that the right and left eyes see the specimen at different angles (see Figure 9). Such microscopes are used most widely where stereoscopic perception facilitates work that must be done on a specimen during observation (biological research, surgical operations on the blood vessels or brain, micrurgy in the eye, and the assembly of miniature devices, such as transistors). The inclusion in the optical diagram of prisms, which act as erecting systems, also serves to facilitate orientation in the field of view. In microscopes of this type the image is not inverted but is erect. Since in stereoscopic microscopes the angle between the optical axes of the objectives is usually ≤ 12°, the numerical aperture does not generally exceed 0.12. The effective magnification of such microscopes is therefore no greater than 120.

Figure 9. Diagram of stereoscopic microscope, which provides three-dimensional perception of observed objects

COMPARISON MICROSCOPES. Comparison microscopes consist of two structurally joined ordinary microscopes with a common eyepiece system. The observer sees the images of two specimens at once in the two halves of the field of view; this enables him to compare the images directly with respect to color, structure, distribution of elements, and other characteristics. Comparison microscopes are widely used in evaluating the quality of treated surfaces and in grading work (comparing with a standard sample). Special microscopes of this type are used in criminology—in particular, to identify the weapon from which a bullet was fired.

TELEVISION MICROSCOPES. Television microscopes operate with a microprojection circuit. The image of the specimen is converted into a sequence of electric signals, which reproduce the image on a magnified scale on the screen of a cathode ray tube (kinescope). Image contrast can be altered and image brightness controlled by purely electronic means—by altering the parameters of the electric circuit through which the signals pass. Electric amplification of the signals makes it possible to project the image on a large screen, whereas ordinary microprojection requires extremely strong illumination, often harmful to the microscopic specimens. The great advantage of television microscopy is that it can be used for studying specimens at a distance, where proximity to the object (radioactive specimens, for example) would be dangerous to the observer.

In many studies, it is important to make a count of the microscopic particles in a specimen (for example, bacteria in colonies, aerosols, particles in colloidal solutions, blood cells) or to determine the areas occupied by the grains of a particular type in sections of an alloy. The conversion of an image into a series of electric signals (impulses) in television microscopes makes it possible to construct automatic microparticle counters that record particles by number of impulses.

MEASURING MICROSCOPES. The function of measuring microscopes is to measure precisely the linear and angular dimensions of the specimens (which are often not at all small). There are two types of measuring microscopes, depending on the method of measurement. The first type is used only when the distance to be measured does not exceed the linear dimensions of the microscope’s field of view. In such microscopes, direct measurement is made not of the object itself but of its image at the focal plane of the eyepiece (by using a scale or a screw-type ocular micrometer). The distance measured on the specimen is then calculated from the known magnification of the objective. The images of specimens are often compared with sample profiles marked on plates of the detachable heads of the eyepieces.

In measuring microscopes of the second type, the stage bearing the specimen and the frame of the microscope can be shifted with respect to each other (most often the stage being shifted with respect to the frame) by using precise adjustment mechanisms. Measuring this shift with a micrometer screw or scale (securely attached to the stage) makes it possible to determine the distance between elements of the specimen.

Measuring microscopes also exist in which measurement is made in only one direction (single-coordinate microscopes). Much more widely used are microscopes in which the stage is moved in two perpendicular directions (the limits being as great as 200 X 500 mm). For special cases, there are microscopes in which measurements (and, consequently, relative stage—frame shifts) are possible in three directions, corresponding to the three axes of rectangular coordinates. Measurements can be made in polar coordinates with some microscopes: the stage can be rotated and is equipped with a scale and vernier for reading the angles of rotation. Glass scales are used in the finest measuring microscopes of the second type; the readings are made with an auxiliary (reading) microscope.

The accuracy of the measurements in microscopes of the second type is much greater than in those of the first type. In the best models, the linear accuracy is usually on the order of 0.001 mm, and the angular accuracy, on the order of 1’. Measuring microscopes of the second type are used extensively in industry (especially in machine building) to measure and monitor the size of machine parts and tools.

In devices designed for especially precise measurements (for example, geodesic and astronomical), readings on the linear scales and graduated dials of goniometric instruments are made with special reading microscopes—scale microscopes and micrometric microscopes. Scale microscopes have an auxiliary glass scale; by adjusting the magnification of the objective, the image of this scale is equalized with the intervals observed between markings on the primary scale (or dial). The position of the division between the marks on the auxiliary scale can then be directly determined with an accuracy of about 0.01 interval between primary scale divisions. The accuracy of the readings in micrometric microscopes, whose eyepieces contain a crosshair or spiral micrometer, is still higher (on the order of 0.0001 mm). The magnification of the objective is controlled so that an integral number of turns (or half-turns) of the micrometer screw corresponds to the shift of the crosshair between the images of the markings on the scale measured.

In addition to the microscopes described, there are a number of more narrowly specialized types of microscopes. Examples include microscopes used for counting and analyzing the tracks of elementary particles and the fragments of nuclear fission in nuclear photographic emulsions; high-temperature microscopes, used to study specimens heated to temperatures on the order of 2000°C; and contact microscopes, used to study the surfaces of living human and animal organs (the objective being pressed against the surface to be studied and the microscope focused by a special built-in system).

In conjunction with other instruments, microscopes are frequently an important component of compound units. Examples include microspectrophotometric units, in which the microscopes are coupled to special monochromators and to devices that measure luminous fluxes; a number of the instruments used in ophthalmology; comparators; and microphotometers.