THE HIDDEN world revealed by a simple microscope has fascinated many an amateur scientist. Over the past 20 years a number of amateurs have become notably proficient at photographing what they see in the instrument. This month I shall review some of the techniques that enable the amateur microscopist to make such photographs.

Information on photomicrography is available from several sources. Mine comes from James Bell of Allston, Mass., whose photographs have appeared in Scientific American and who now supplies examples (reproduced on the next two pages) of what his photomicrographic techniques can achieve. The variety of specimens you can examine and photograph is almost endless, and so Bell's examples are meant only as an introduction. Although professional photographers often work with elaborate and expensive equipment, Bell's photographs show that even with fairly simple and inexpensive equipment you can make strikingly beautiful and scientifically revealing photographs.

Bell worked with a monocular Bausch and Lomb microscope made in 1915. It was fitted with old Leitz objectives that had magnifying powers of 10 and 100 and with eyepieces that had magnifying powers of 10, 7 and 2.5. He also had a mirror to direct light into a condenser that caused the light to converge on the specimen. He removed the lens from his 35-millimeter Asahiflex camera and attached the camera to the microscope with a commercial lighttight microscope adapter. He did his focusing on the ground-glass back of the camera. To avoid jarring the camera during his exposures Bell relied on a cable release. He made the photographs at shutter speeds between 1/500 and 1/25 second and with Ektachrome-X or Ektachrome-64 film.

Intense light is needed because only a small fraction of it reaches the film. Bell got his light from an ordinary slide projector with a 400-watt bulb. To collimate the light he replaced the lens of the projector with another light condenser. If you work with less light than Bell did you would have to increase the exposure time by a second or more. Clear photographs would be more difficult to obtain; with moving specimens you would be likely to get only a blur. If the light was too bright for a particular photograph, Bell put one or more neutral density filters in the light path from the projector. Such filters can be bought from the Edmund Scientific Company (7875 Edscorp Building, Barrington, N.J. 08007) or made by photographing a sheet of white paper and developing the negative.

The infrared component of the light beam may be harmful to the specimens, particularly if they are living organisms. You can remove it from the beam by placing a container of water (the sides should be parallel to avoid unwanted optical effects) or several pieces of optical glass in the light path. A projector may have a built-in heat sink that would serve the same purpose.

Bright-field illumination is the easiest of several kinds of illumination to use but is quite limited in revealing details of the specimen. Bell achieves this kind of illumination by angling his mirror so that the substage condenser (the one below- the specimen holder) causes the light to converge on the specimen. When you do this, you see the light transmitted through the specimen. The technique therefore does not work with thick or opaque specimens and does not always show the internal structure of thin specimens. Moreover, the natural colors of a specimen are often lost in the bright light. Still the technique may be useful in black-and-white photography of thin specimens.

To obtain the highest possible resolution, which is crucial in high-power work, you should follow the directions for Köhler illumination in Photography Through the Microscope, the bible of photomicroscopy published by the Eastman Kodak Company. In essence the procedure calls for you to adjust the mirror, the collector (the lens between the mirror and the light source) and the condenser so that the filament of the lamp is focused on the condenser and the condenser focuses an image of the collector onto the specimen holder. With this adjustment the specimen is evenly illuminated and you do not see a focused image of the filament.

The first of Bell's photographs, which is at the top left above, is an example of the bright-field technique. Bell used a magnification of 1,100 to photograph living cells of the freshwater alga Elodea to reveal the individual chloroplasts. Moments after the photograph was made the chloroplasts began to circle in the cells as they engaged in photosynthesis in the light from the projector. Although details appear in the photograph, the image is not as sharp as it is in the other photographs, partly because the high magnification reduced the depth of field.

You can get good contrast in your photographs by putting the specimen on a dark background. To create such a background while still illuminating the specimen Bell placed a black dot on a clear glass filter that he slipped into the filter holder just below the substage condenser. The diaphragm of the condenser was opened either completely or almost so to provide a wide cone of light. The purpose of the black dot is to cast a shadow against which the specimen is to be photographed. The light leaking around the edge of the dot illuminates the specimen in white light so that its natural colors are still seen. In the absence of a specimen the crossing light rays would reach the objective of the microscope so diverged that no light would enter the tube, and you and your camera would see only a dark field. Once the specimen is in place, however, light scatters from it into the tube, enabling you to see the specimen. This technique obviously requires a strong light source because only a small fraction of the initial light will reach the camera.

The dark-field filter is made by gluing a small opaque circle of black construction paper on a transparent glass filter. The circle should be about five millimeters in diameter or somewhat larger. Determine the size by trying it in your microscope. The field should be dark when no specimen is in the holder but properly illuminated when a specimen is there. Instructions for determining the size were given in this department some years ago [April, 1968], along with much other information on photomicroscopy.

Bell has been able to use dark-field illumination with only his lower-power objective (10X). Greater magnification produces photographs with less contrast because of the difficulty of keeping the objective lens in the dark cone cast by the opaque dot. Objectives of higher power must be brought closer to the specimen for proper focusing, and then the lens begins to intercept some of the light rays coming around the opaque dot. To go to greater magnifications with the technique Bell would have to buy a dark-field condenser that provides a finer cone of darkness.

With the dark-field technique Bell photographed a specimen of hydra and some volvox colonies. He found these specimens in local ponds, as you can. Descriptions of the rich array of specimens available are given in a number of books of the kind you can buy in a natural-history museum. My favorites are the two children's classics by Richard Headstrom listed in the bibliography for this issue [below].

Since Bell's kind of photography can be done with live specimens, your photographs (if you are patient enough) may catch the organisms in various types of characteristic behavior. For example, you can photograph a hydra eating. By feeding it small aquatic worms or tiny chunks of raw beef you can induce it to open its mouth and can see how it uses its tentacles. Headstrom notes that with a small amount of acetic acid or methyl green you can provoke the hydra to discharge one of its nematocysts. These barbed tubes are fired with such force that they can penetrate the animal's prey.

Volvox is a gelatinous spherical colony made up of hundreds of flagellate organisms. If you look at a colony closely, you will find that the surface is embroidered with flagella, the whiplike appendages by which individual flagellates propel themselves. Headstrom recommends collecting specimens of this kind with a glass tube. Once you find a specimen in a pond (perhaps with the aid of a magnifying glass) lower a tube into the water while keeping a finger over the top of the tube. Then remove the finger. The specimen will be drawn up into the tube by an influx of water. Replace your finger to retain the specimen and the water in the tube. Carefully release the water over a microscope slide until you have the specimens on it. The best slides are the ones with a concave well to hold the specimen and a little water.

Some specimens contrast better with a colored background than with a dark one. To accomplish this coloring (called partial Rheinberg illumination) Bell replaced the opaque black dot with a transparent colored dot. For example, to provide contrast for a red specimen he would use a blue or green dot cut from colored plastic filters of the type available from Edmund Scientific. The specimen continues to appear in its natural color, since it is still illuminated primarily by the white light leaking around the dot.

A blue background served in Bell's photographs of the crystals of an ester of cholesterol that he had recrystallized under a glass cover slip. The background enables you to see the interfaces between the crystals and the cracks caused by stress inside individual crystals. To reveal some freshwater-snail embryos still within the egg, Bell made the photograph with a green dot at 80X. This delicate detail would have been totally lost in bright-field illumination.

With full Rheinberg illumination Bell can select any color to illuminate the specimen and can choose a different color for the background. The only change in the procedure is to glue a ring of lightly colored plastic around the darker dot. Then the light transmitted through the dot gives the background color as the light coming through the ring colors the specimen.

Bell photographed the colonial rotifer Conochilus with a blue dot and a yellow ring at 120X. He pointed out in a letter to me that the rotifers would have been almost invisible in bright-field illumination; the color contrasts are essential in order to distinguish their edges and internal structure.

Bell finds that the most effective technique for adding color contrast to his photographs is the use of polarized light. The technique works, however, only with birefringent materials such as crystals, muscle and certain small multicellular organisms. A polarizing filter is inserted in the slide projector (just as an ordinary slide would be) to provide linearly polarized light. A second filter, put on the eyepiece of the microscope, can then be turned until the field of view through the microscope is either dark or bright as the filter either blocks or transmits the sense of polarization coming through the instrument.

When a birefringent material is inserted in the specimen holder, the polarized light produced by the first filter is altered in polarization when it passes through the material. Appropriately altered some of the light can then pass through the second polarizing filter above the eyepiece even though the filter would otherwise block the light. (The details of how the sense of polarization is changed by the birefringent material were covered in this department in December, 1977.) In essence the light is transmitted through the material when part of the light is polarized along one axis and another part is polarized along a perpendicular axis. Both axes are perpendicular to the light ray. The velocity of light is higher for one of the senses of polarization than it is for the other. A result of this difference is that the two senses of polarization can emerge from the material out of step. Since the emerging polarization is determined by the combination of the two senses of polarization, the emerging light could be polarized in several ways.

If the two senses of polarization emerge out of step by half a wavelength, the emerging light is again linearly polarized, but now along an axis perpendicular to the polarization sense of the incident light. This emerging light might be transmitted or blocked by the polarizing filter at the eyepiece, depending on the orientation of the filter.

If the birefringence has forced the emerging senses to be a quarter of a wavelength out of step, the emerging light is elliptically polarized, which means that the tip of the polarization vector rotates about the light ray, mapping out an ellipse. This light will be passed by the filter at the eyepiece regardless of the filter's orientation. If the emerging senses of polarization are in step, the emerging light is polarized in the same way that the incident light is. The orientation of the second filter will then determine whether or not you see any light.

Which of these results occurs depends on the thickness and birefringence of the specimen and on the wavelength of the light. For a particular area of the specimen the red end of the white light incident on the specimen may end up being blocked by the second polarizing filter whereas the blue may be transmitted. That area would appear blue to you. Another area may be colored yellow be cause the other colors of the incident white light are blocked. The advantage in using polarized light in this way is that the color variation produced in an otherwise colorless specimen enables you to distinguish details and internal structure in the specimen.

Bell sent along two photographs made with polarized light. The first one (at 120X) shows hippuric acid crystals that had been dissolved in isopropanol and then allowed to recrystallize on the slide. The sample was sufficiently birefringent and nonuniform to produce many colors. In his other example (at 150X) resorcinol crystals were dissolved in acetone, which was then burned off to facilitate quick recrystallization.

Further color contrast can be produced by inserting one piece or more of clear cellophane or transparent tape in the filter holder below the specimen. Both are birefringent materials that alter the polarization of the light. Instead of cellophane you can use a thin sheet of mica, which can be easily split off from thicker layers of mica with a razor blade. Either material is described as a retardation filter because it forces one sense of polarization to lag behind the perpendicular sense. A retardation filter is useful in enhancing the color variation in a weakly birefringent specimen. Not all kinds of cellophane work, and you will have to experiment to find a kind that does. You could also build up layers of stretched plastic food wrap, following the procedure I gave in my article on birefringence.

Bell has submitted several other examples of his use of polarized light. A living water flea with its young in a brood pouch was photographed at l00X with a blue dot to provide background and with cellophane and polarizing filters to enhance the internal structure of the animal. The eyespot is left black whereas the digestive tract is made orange.

The copepod Cyclops was photographed (at 50X) in a similar way except that a black dot was employed to give a dark background. The animal appears with its eyespot red, two muscle bands yellow and its digestive tract brown. The two pear-shaped sacs on the sides contain eggs. A great deal of this detail would be lost in simple bright-field illumination.

The effect of cellophane can be seen in two of Bell's photographs, both of which are of ascorbic acid (vitamin C) that he recrystallized. The photographs show the same two adjacent crystals and were made identically except that cellophane was used in the second photograph. Notice the additional color due to the birefringence of the cellophane.

Bell prepared the crystals by dissolving the ascorbic acid in isopropanol, putting some of the solution on a microscope slide and then burning off the alcohol. The crystals formed within a few seconds. After one set of photographs has been made he can redissolve the ascorbic acid and start over. The technique yields crystals thin enough to be transparent and therefore readily photographed. An alternative method of forming crystals is to allow the solvent to slowly evaporate.

Another way to make crystals is to melt the substance on a glass slide over a flame. It will crystallize as it cools. Bell does not favor this technique because the melting point of some substances is too high and he invariably ends up breaking too many slides. He does point out that if you want to study certain kinds of liquid crystal, you will have to use the technique, cooling the substance slowly from its melting point in order to get the liquid crystalline state. Bell's photograph of a nematic liquid crystal was made at 100 X at room temperature after he had dissolved the material in acetone. The motion in this recrystallization was so rapid that he had to shoot the photograph at 1/500 second to stop the activity.

Some specimens are too thick to be photographed with transmitted light and instead call for reflected light. To accomplish this Bell props his projector up on books and adjusts the angle of the incident light until he gets the right illumination on the specimen. An example of the result is his photograph of a butterfly wing. Many of the colors you see in butterfly wings, on the back of beetles and in bird feathers are due not to pigmentation but to the interference of light waves. Some butterfly wings consist of layers of transparent cuticle. When light is reflected from one of the layers, some of it comes from the top surface and some of it comes from the back surface after passing through the layer. The two emerging rays can interfere with each other to produce colors, just as they do in thin soap films. (I described such interference in this department last September.) You might want to try several different kinds of butterfly wing. They can be ordered from Gilmour-Vendco, Inc. (12685 Highway Nine, Box 196, Boulder Creek, Calif. 95006), or bought at gift shops in many cities. The interference colors would be weak if the wing were illuminated only with transmitted light. Then the colors would have to arise from the interference between light transmitted through the cuticlelike layer (with no internal reflection) and light reflected twice inside the layer before it emerges. This light would be so much weaker than the unreflected light that little interference (and hence little color) would result.

To find the correct exposure you can either bracket your shots with a range of exposure times or attach a light meter to the microscope. Bell employs a homemade light meter featuring a digital millivolt meter and a silicon photocell. He has mounted the cell in a cardboard housing that fits snugly over the viewfinder of the camera. As the light intensity varies with the specimen and the mode of illumination, the resistance of the photocell varies inversely. Through intermediate circuitry these variations change the readout on the millivolt meter. With some experimentation Bell was able to construct a table of readouts so that he could immediately interpret them in terms of the proper shutter speed. (A design for a simple but highly sensitive light meter employing a cadmium sulfide photoresistor appeared in Popular Electronics for June, 1977.)

If you want good reproduction of the natural colors of a specimen, you must be careful about the type of color film you use: it must be matched in color temperature to your lamp. All the lamps look white to the human eye, but the actual distribution of intensities across the visible spectrum will depend on the temperature of the emitting surface in the lamp. A low-temperature lamp of white light has fewer of the low frequencies of visible light (that is, the blue end of the spectrum) than a lamp of higher temperature. The various color films have been adjusted somewhat to compensate for this difference in the color distribution of white light. Nevertheless, there may be some discrepancy between the true colors of a specimen and what you see in a color photograph. You can remedy the situation by putting a color filter in front of the projector (or whatever light source you use). The Kodak book explains which filters should be employed.

Bell has a 400-watt DAT lamp in his projector. The temperature of the emitting filament surface is so high that the light is similar to sunlight in its color distribution. Bell therefore uses daylight color film instead of film that is color-balanced for tungsten lamps operating at a lower surface temperature.

Teresa Owens, as an undergraduate at Reed College in Portland, Ore., has done some careful (and exhausting) work on the rate of cooling of water from various initial temperatures. As I said in this department for September, 1977, hot water will sometimes reach the freezing point before initially cooler water does. The experiment is so rich in variables that verifying the effect and tracking down its cause are challenging.

With an experimental setup similar to mine Owens monitored the temperature of the freezer environment and the temperature of several places in a beaker of water placed in the freezer. Her results indicate that the effect is probably present but can easily be lost in the variation of the environmental temperature between experimental runs. From her data she concludes that the two most important factors determining the effect are the initial environmental temperature and the circulation of air over the top of the container. She believes the temperature gradient inside the water and the mass lost to evaporation are not as important. Much more work could be done on this experiment. If you pursue it, please let me know what you find.

Bibliography

PHOTOGRAPHY THROUGH THE MICROSCOPE. Eastman Kodak Company, 1974.

ADVENTURES WITH A MICROSCOPE. Richard Headstrom. Dover Publications, Inc., 1977.

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