Synthetic images of many objects can be made by manipulating the light so that rays transmitted by

the specimen emerge altered in intensity, direction or color or some combination of the three. The principal scene of these operations is the substage of the microscope, which includes the condensing lenses and the iris diaphragm associated with them, a filter, a mirror and a lamp assembly. Control of the image is largely lost after the rays emerge from the specimen. The body-tube assembly of the instrument, which includes the objective lens and the eyepiece, merely magnifies the image to a size that is convenient for viewing. The performance of this part of the microscope is fixed by the instrument maker. The substage components provide opportunities for innovation in creating images; the opportunities have sustained the interest of amateurs in microscopy for more than a century. Some techniques of lighting that yield colored images of remarkable detail are discussed by Mortimer Abramowitz, assistant superintendent for instruction in the public schools of Great Neck, N.Y. He writes:

"I have been experimenting with two systems of lighting. One was first suggested by the British microscopist Julius Rheinberg. The second makes use of polarized light. Both systems add color to images that would otherwise appear in black and white.

"I am primarily interested in making color photomicrographs. For this reason I begin experiments by adjusting the substage components of the microscope for a type of lighting that was developed specifically for photomicrography by the German microscopist August Kohler. Kohler illumination is then modified for either Rheinberg illumination or polarized light. In Rheinberg illumination color is introduced by inserting filters of selected hues into one or more regions of the light that floods the specimen. Alternatively, color effects can be developed in Kohler illumination by inserting polarizing filters of two kinds at appropriate points in the optical path of the instrument.

"The kind of specimen used for demonstrating Rheinberg illumination should be one with prominent features. Examples include the appendages of a small insect, a thin section of wood and living protozoa. Magnification of about 100 diameters, such as is provided by a 10X objective lens in combination with a 10X eyepiece, is adequate. Open the iris diaphragm of the condenser fully. Turn on the lamp and adjust the mirror to direct some light through the instrument.

"Kohler illumination is established by a series of simple adjustments. Move the body tube up or down until the specimen appears in sharp focus. Partly close the diaphragm of the lamp and move the substage condenser up or down until a sharp image of the diaphragm appears in the plane of the specimen, as viewed through the eyepiece. Center the light by moving the lamp or mirror.

"Enlarge the diameter of the disk of light by opening the lamp diaphragm until its edge just disappears from view. Remove the eyepiece, observe the spot of light that appears in the rear lens of the objective and close the iris diaphragm of the substage condenser until the diameter of the spot appears to be about three-quarters of the diameter of the rear lens. Replace the eyepiece and examine the specimen. It will appear against a bright background. Most details will seem darker than the background, but (depending on the nature of the specimen) some may seem brighter. In general the brightness of the background will be uniform.

"The microscope has now been adjusted for 'bright field' work. The term refers to a form of lighting that is useful for making photomicrographs of stained specimens in black and white or color. The tenuous structures of some specimens, however, are difficult to see against the bright background. Many of the structures can be brought into view by a simple modification of Kohler illumination. The effect of the change is to reverse the character of the scene: the image appears bright against a dark background.

"This effect can be achieved by blocking the solid cone of rays that normally enters the objective lens and illuminating the specimen with the surrounding hollow cone of rays that remains. The solid cone is blocked by putting an opaque disk of appropriate diameter in the center of the substage iris diaphragm, which is just below the condensing lenses. The opaque disk can be cut from black paper. It is cemented to a disk of clear glass that fits into the filter holder of the instrument's substage.

"The unobstructed circular space that remains between the edge of the opaque disk and the edge of the filter holder transmits a hollow cone of light through the condenser. The light illuminates the specimen with oblique rays that miss the objective lens. None of the rays reach the eye directly. The field appears dark. Features of the specimen, however, diffract, or scatter, light from the hollow cone. Some of the rays enter the objective lens. They are brought to focus at the eye in the form of a bright image that appears against the dark background. Minute details that are easily overlooked in a bright field stand out clearly against the dark field in an image of high visibility.

"To prepare an opaque disk of the right size for dark-field work, adjust the instrument for Kohler illumination. Remove the eyepiece and replace it with a pinhole aperture. The pinhole, which should be about a millimeter in diameter, can be made in a thin sheet of black cardboard or metal that is painted flat black. The hole must be centered on the axis of the body tube. It can be supported in this position by an opaque bottle cap that fits snugly into the body tube. A larger hole is cut in the center of the cap to expose the pinhole [below left].

"With the pinhole in place and Kohler illumination established, look through the pinhole and close the iris of the condenser until the edge of the iris barely cuts into the field of view. Open the iris until its edge just disappears. Measure the diameter of the opening of the substage iris diaphragm at this adjustment An opaque disk about 5 percent larger in diameter will, when it is placed in the substage filter holder, block all direct rays to the objective lens and result in dark-field illumination.

"Although dark-field illumination reveals many features of specimens that cannot be seen clearly against a bright field, visibility can be increased still more by introducing color into the image. The background can be made to appear in color by substituting a transparent colored disk for the opaque disk. Specimens then scatter white light into the objective and appear bright against a background of the selected color [see Figure 7].

"What I have described is a simple version of Rheinberg illumination. Full Rheinberg illumination is established by installing a colored filter in the form of a ring in the clear space between the colored stop and the edge of the filter holder. When complementary hues are selected for the colored stop and the surrounding annulus, the specimen appears in one color against a background of contrasting color. Effective combinations are red against green and blue against yellow. A third color can be introduced by dividing the annulus into four equal parts and installing contrasting colors in adjacent sectors-red, say, in one pair of opposite quadrants and green in the other pair. The colored stop, which might be dark blue, provides the third color [see Figure 10].

"The colored field is lighted by the solid cone of direct rays from the colored stop, whereas the image is formed by annular rays of lesser intensity that are scattered into the objective by diffraction. If the two Rheinberg filters are equal in density, the rather dim image of the specimen may be lost in the brilliant background. The relative intensity can be adjusted in several ways. The density of the colored stop can be increased by means of multiple layers of colored plastic. Alternatively, a neutral-density filter can be combined with the colored plastic. I cut such filters from a piece of developed negative film that has been exposed in a camera to a sheet of white paper. The density of the negative is controlled by the exposure.

"The brightness of the background color can be made continuously variable by the use of two Polaroid filters. The filters transmit waves of light that vibrate in a single plane but absorb waves that vibrate in other planes. A filter consists of a plastic sheet in which needle-like crystals of iodoquinine are embedded. They all point in a single direction, like the pickets of a fence. Light waves that vibrate in a plane parallel to the crystals are transmitted by the Polaroid. Waves that vibrate in other directions are increasingly absorbed by the filter as the plane of vibration departs from the direction in which the crystals point.

"Light that makes its way through one sheet of Polaroid will be transmitted by a second sheet turned so that the crystals of both sheets are parallel. Conversely, the second sheet will largely absorb light transmitted by the first if its crystals make a right angle with respect to those in the first sheet. By placing one Polaroid filter in the central stop of the Rheinberg filter and a second one in or on the eyepiece of the microscope, the brilliance of the background can be controlled by rotating the upper Polaroid filter. Polaroid filters, sheets of plastic in assorted colors (for making Rheinberg filters), clear disks of glass, Canada balsam and related supplies for the microscope are available from the Edmund Scientific Co., Barrington, N.J. 08007.

"Three-color Rheinberg illumination is particularly effective for examining specimens that display recurring features.

These specimens include textiles and the fossils of marine plants such as diatoms. When a specimen of fine silk is examined under three-color Rheinberg illumination, the warp appears in one color and the woof in another; both appear against the background of the third color. The colors can be blended and interchanged by rotating the specimen.

"Color filters for Rheinberg illumination can be made by cutting the central disks and the surrounding rings from thin sheets of colored plastic and cementing them with Canada balsam to disks of clear glass that fit into the filter holder of the instrument. For best results the edges of adjacent colors should be covered by an opaque border about three millimeters wide. The border can consist of a ring of black paper cemented directly to the filters. The accompanying photomicrographs [top] illustrate some effects of combining two and three colors. Most of my photomicrographs are made with a Nikon S-Ke microscope and a Nikon EFM Microflex camera.

"Images so colored do not correspond perfectly to the actual appearance of the object under examination. The same situation is more or less true of all microscope images. Complete correspondence between the object and its image can be approached but not attained because light from a point comes to a focus as a small disk of finite size. Disks resulting from closely spaced points merge into a fuzzy spot. Moreover, all the light from a point would be required for the formation of a perfect image of the point, whereas only a small portion of the emitted light ever reaches the image. Most of the light is scattered in other directions. For such reasons the microscopist must judge the true nature of the object by examining the image while taking account of the techniques that were used to create it. Color, when employed in this sense, becomes a powerful aid for isolating individual details in specimens.

"Although Rheinberg illumination increases the visibility of specimens that have physically distinct parts, it is of less use for examining the fine, transparent features of rocks, crystals and plant fibers and of no use for detecting patterns of stress in glass and other amorphous substances. Many of these materials, however, have an optical property of great value to the microscopist. They rotate the plane in which light waves vibrate. This property can be used for creating color in many features of otherwise clear and transparent materials.

"The technique requires only two accessories: a pair of Polaroid filters and a retardation step wedge, which consists of a series of progressively shorter strips of Scotch tape applied to a microscope slide [left]. The material to be examined is placed on the slide of the microscope, lighted by the Kohler technique and focused. A Polaroid filter is placed in the filter holder below the substage condenser. The second Polaroid filter, which is called the analyzer, can be placed on top of the eyepiece and supported in that position by a holder made from an opaque bottle cap. The analyzer can also be put in a holder attached to the front end of the eyepiece. When the analyzer is rotated, the brightness of the background will vary from a maximum to a minimum twice during each revolution. The brightness of details in the specimen will vary similarly and some may appear in color.

"If the retardation step wedge is now inserted under the specimen in a position that admits light through two thicknesses of Scotch tape, the image will be seen as a spectacular pattern of hues that span the spectrum. The various colors can be made to blend, to shift from one detail of the specimen to another and to change in saturation by rotating the wedge with respect to the optical axis of the microscope. Rotate only the wedge, not the specimen slide. The insertion of the wedge will move the specimen and throw the image out of focus. The instrument should be refocused each time the position of the wedge is changed. The saturation of the colors can be decreased from vivid hues to pale pastels by advancing the wedge so that the light passes through several additional layers of Scotch tape.

"More pleasing colors and images of improved resolution can be produced by substituting thin sheets of mica for Scotch tape. The sheets can be split from a block of mica by thrusting the point of a sewing needle into the edge of the block. The optimum thickness of the sheets must be determined experimentally but will be about .003 inch. Split off a sheet about the thickness of tissue paper and try it on the stage of the microscope. If the colors are brighter than those that appear when Scotch tape is used, cut the sheet into strips and make the wedge. If the colors are not brighter, try thinner or thicker sheets.

"Some transparent materials fail to show color when they are examined by this technique. Indeed, nonopaque solids can be divided into two groups according to how they appear when they are examined between a pair of Polaroid filters that are rotated to the 'crossed' position at which they transmit minimum light. One of the two groups consists of substances that do not rotate the plane in which light vibrates. Such substances, which are described as being optically isotropic, remain dark when viewed between the filters; an example is ordinary table salt. The second group consists of substances that do rotate the plane of polarization. Called anisotropic substances, they include such readily available chemicals as potassium chlorate, oxalic acid, calcium carbonate, boric acid, salol and DDT. Many plant fibers, most transparent plastics, thin sections of rock and also glass that has been heated until it is soft and cooled without annealing are anisotropic.

"Specimen slides of these materials, particularly the chemicals, are easy to prepare Place one or two drops of distilled water in the center of a clean slide and sprinkle a few crystals of the chemical into the water. Granulated sugar is an excellent specimen, as is vitamin C. Do not add more of the substance than will dissolve in the water at room temperature. Place the slide in a dustproof box until the water evaporates. Examine the resulting deposit of crystals with polarized light. (Some chemicals that do not dissolve in water can be melted by heat and cooled on a slide, where they recrystallize.)

"Most of the accompanying photomicrographs were made with a 35-millimeter camera equipped with a self-contained exposure meter and a telescopic eyepiece through which the image can be viewed until the moment of exposure. Pictures of comparable quality can also be made with an improvised lightproof box for supporting the film [see "The Amateur Scientist," February, 1961]. In either case the illumination of the image is fairly low because much of the light is absorbed by the polarizing filters. The experimenter can compensate for part of the absorption by increasing the brightness of the lamp. The filters are sensitive to heat, however, and may be damaged by temperatures above 60 degrees centigrade. They can be protected by the insertion of a heat filter between the lamp and the lower Polaroid. Heat filters of this type are used in 35-millimeter slide projectors and are available from dealers in photographic supplies.

"Exposures can be made with flashbulbs if care is taken to put the bulb in the position normally occupied by the microscope lamp. If the flash results in overexposure, the intensity can be reduced by inserting a neutral-density filter between the flashbulb and the mirror of the microscope The neutral-density filter must be of good optical quality to prevent the introduction of spurious color.

"A light meter sensitive to .5 footcandle and capable of indicating exposure intervals of 20 seconds or more is helpful for determining exposures. I usually 'bracket' the meter reading by making exposures one stop higher and one stop lower than the ones indicated by the meter. With practice one learns to judge the brightness of the image by eye and to make the exposure accordingly. Color film is sensitive to the length of the exposure as well as to the intensity of the light.

"Some films take on false color during exposures of more than 1/10 second. This error can be corrected by the use of a special filter. For example, when exposing Kodachrome IIA for intervals between 1/10 second and 10 seconds, insert a CC lOR filter between the lamp and the microscope; for exposures between 10 and 20 seconds use a CC 20R filter. Incidentally, optical imperfections in filters degrade the quality of the image least when they are placed between the light source and the instrument; the degradation is somewhat greater when they are placed between the objective lens and the camera. Filters should never be used between the specimen and the objective lens.

"The hues of color photographs are determined in part by the temperature of the light source. Kodachrome IIA is designed for a temperature of 3,400 degrees Kelvin. When this film is exposed by a lamp that is rated at 3,200 degrees K., for example, a correction filter must be used, in this case an 82A Wratten filter.

"Full information on-the characteristics of the various films and the conditions under which compensating filters must be used can be found in data books or in the leaflets that accompany the films. I have had excellent results with Polacolor film. I use the four-by-five-inch film sheets in a film holder that is attached to the eyepiece fixture of the camera. When using this material, I find that exposures are somewhat more difficult to estimate. The fidelity of the prints is not quite up to that of positive transparencies, but the overall results have been gratifying."

Bibliography

HOW TO USE A MICROSCOPE. W. G. Hartley. American edition by John J. Lee and Bernard Friedman. The Natural History Press, 1964.

PHOTOGRAPHY THROUGH THE MICROSCOPE. Eastman Kodak Company, 1962.

PRACTICAL USE OF THE MICROSCOPE INCLUDING PHOTOMICROGRAPHY. George Herbert Needham. Blackwell Scientific Publications, 1958.

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