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Photomicrography

Photomicrography is the correct term for photography of tiny objects using a microscope. Some older works on microscopy use microphotography, but this term has come to be associated with tiny photographs such as microfiche. A photomicroscope is the kind of microscope used for photomicrography.

Attachment cameras and SLRs

Any microscope can easily be converted to a photomicroscope. The most straightforward adaptation uses a photomicrographic attachment camera. These cameras usually have a beam splitter with a camera on top and an eyepiece on the side so that you can see exactly what will be photographed. These are rather expensive; you can buy a single lens reflex camera (SLR) body for about one tenth the price. The SLR is a bit harder to work with, but will produce quality photomicrographs.

The maker of the microscope or a camera shop can provide an adapter tube to fit over the body tube and eyepiece. The eyepiece replaces the camera lens. The adapter tube has threads on the top that fit a T adapter, which the maker of the SLR provides. The lens is removed from the SLR, and the T adapter is attached. The SLR with T adapter is screwed to the adapter tube, and the conversion is complete. Most SLRs connect to the T adapter with a bayonet mount, and can be clipped on and off the microscope in a matter of seconds.

In the case of a binocular microscope, one of the eye tubes can be used as the camera mount. Some microscopes have a special photo tube with a beam splitter to send part of the light to the camera. Check with the manufacturer for special attachments that may be needed.

The SLR camera body should have a manual mode and the ability to use aperture priority when in automatic mode. Without aperture priority, the exposure gadgetry shuts down when the camera lens is removed. We want the exposure meter to work when a microscope eyepiece is being used as the lens.

Considerable time will be spent looking into the SLR view finder. A clear view screen with cross hairs makes viewing dim images easier than does a matte screen. Most SLRs have a matte screen surrounding a small, clear, central screen that is either split into two hemispheres or that has cross hairs. For really critical focus at high magnification, use the clear center. The matte screen can be changed out on some cameras. On other cameras, magnifiers are available to fit onto the view finder, or a hand magnifier can be used to make viewing the clear center of the screen easier. (If the clear part of the screen is split into hemispheres, when the SLR is connected to a microscope only one of the two will be clear -- the other will be black. By moving your eye, the clear and black hemispheres will switch.) When you think that you have the image in focus, slightly rock your eye back and forth across the plane above the view screen. If the part of the image that you wish to have in focus does not move in relation to the view screen cross hairs or within the hemisphere, it is in focus.

The view screen of SLR cameras should be covered before the cable release is pressed if you are using the exposure meter. Stray light in the view finder affects exposure metering.

Fixed lens cameras

Cameras with fixed lenses can also be used for photomicrography. They are more difficult to set up for use than SLR cameras, and the quality of the photomicrographs that they make is usually inferior. This is because the extra lens introduced into the system is not needed. However, you may already have one of these cameras on hand.

The fixed lens camera must be mounted above the eyepiece, and the camera mount must be sturdy. You can use an enlarger frame or a tripod, or you can build a mount. Turn on the microscope lamp and move a piece of paper up and down above the eyepiece. The spot of light on the paper will become smaller and then larger. The point where the light is smallest is the eyepoint. The eyepoint must be located at the center of the front lens of the camera. If the lens is not in the right position above the eyepiece, aberration will be introduced. The camera lens, if it allows focusing, should be set at infinity and the camera aperture should be completely open. A separate light meter often has to be used, and the fixed lens camera should allow manual exposure timing.

Focus on the specimen before swinging the fixed lens camera into place. Since eyes vary, the following method will help to insure that the focus is right for the camera. Using binoculars or a telescope, focus on an object on the horizon (or at least several hundred feet away). It is helpful if the telescope has a graticule to use in focusing, but not strictly necessary because the telescope will still restrict your eye's ability to accommodate. Now look through the telescope at the eyepoint while focusing the microscope. This will help to assure that your eyes do not accommodate while focusing. If you are still getting fuzzy photographs, make a careful record of some test shots with the camera lens set differently each time.

Just before taking the shot, swing the camera around to the eyepoint, throw a black cloth over the camera and eyepiece to exclude external light, and take the shot. A tube can also be fitted between the camera and eyepiece.

Instant cameras

Instant cameras for microscopes are also available. Both the camera and its film are expensive, and the choice of films is restricted. These cameras are also rather large mechanisms that are inconvenient to use on some microscopes without photo tubes. Quick access to results may override these disadvantages in a particular application.

Vibration

Vibration is a problem in photomicrography, the slightest vibration causing fuzzy photos. Always use a cable release, and place the microscope in a location that receives no vibration. It may help to set the microscope on vibration damping material. A few SLR cameras allow you to lock up the mirror before pressing the shutter release. The movement of the mirror gives the SLR camera its characteristic 'thunk' when the shutter is released. If your camera does not allow mirror locking (most do not), attention to other vibration damping methods should work. Longer exposure times will also minimize the problems caused by vibration. You can block light from the condenser with a card, open the camera shutter, remove the card for the exposure, replace the card, and close the shutter. No vibration at all will occur.

Exposure

Start experimenting with the simplest setups possible. This allows you to make some predictions about more complex setups. Use black-and-white film, brightfield illumination, only the filters specified by the film literature, and a specimen of the type that will most often be photographed. After a base of information is established, move on to color film, special filters, special illumination setups, and unusual specimens. Do not be discouraged by this stepped approach; you will probably begin taking useful photographs almost immediately.

An exposure meter cannot do its job when you are photographing a small object that contrasts with a large background. It makes no difference whether the background is light or dark, the meter will not make a correct exposure of the object. This is a problem common to all photography that is compounded in photomicrography because it is not possible to take a reading of a part of a microscopic specimen without specialized equipment.

Your camera will need a manual mode. You will purposely overexpose or underexpose the shot as a whole so that the object of interest is exposed correctly. Taking a number of shots at graduated shutter timings is called bracketing. To do this, take a test exposure at each manual setting of the camera and keep complete records of the illumination setup used, of the amount exposure, and of the characteristics of the specimen. Tell the film processor that the film consists of experimental shots, and have all exposures developed and returned in the sequence that they were taken. When the prints are returned, compare them to your notes.

Film

Whether you are working in color or black-and-white, you should generally choose professional film with fine grain. High contrast film is often required for unstained specimens. Professional grade films have a short shelf life and should be kept refrigerated when not in the camera. Keep film tightly sealed in a plastic bag so that it does not become moist or contaminated while in the refrigerator. If short exposure times are required, you will have to make the compromise of faster film speed for coarser images.

Slide film gives better rendition of color than print film. You will still be able to get color prints made from the slides. Since any particular color film is made for use at a particular color temperature (see p. 91), you will have to consider the color of your illuminator and make corrections using filters suggested by the literature that comes with the film. Because of low light and long exposure times, you will often have to deal with reciprocity effect. Contrast of black-and-white film and color balance of color film is affected by long exposure times. Literature that comes with the film will also suggest remedies for this problem. A good starting collection of filters for color work will include 82A and 82C color balancing filters, an 80A daylight conversion filter, and neutral density filters. The neutral density filters are needed for color work because the color of the light will change if the light is dimmed. Green, red, yellow, and blue filters are useful for both color and black-and-white work.

Field Curvature

If the microscope does not have plan optics, then either the center or the edges of a photograph taken through the microscope will be out of focus. This is true even though you may not have noticed this when simply looking into the microscope. Many errors that your eye will forgive the camera will not.

Try the following fixes for field curvature:
1) The photographs can be cropped to exclude the edges.
2) Try using a higher power eyepiece, which will show less of the edges of the field imaged by the objective.
3) The aperture and field diaphragm must be set carefully to enhance depth of field; remember that depth of field increases as NA decreases. If you are using brightfield illumination, take pains to bring your microscope as close as possible to perfect Köhler illumination before taking a photomicrograph. This gives as much depth of field as possible without loss of resolution.
4) The camera can be moved away from the eyepiece so that only the center of the field is recorded. Most adapters for SLR cameras will slide up and down the body tube. Choose the position that places the film plane as far away from the eyepiece as possible. Cameras with fixed focus lenses can be raised a bit above the eyepoint. Do not move either kind of camera so far away from the eyepiece that empty magnification is introduced into the final image.

Photographing Specimens that Move

Specimens that move can be a real problem. The best thing to do is restrict their movement or incapacitate them. A tiny animal, placed under a cover slip in a drop of water, will become incapacitated as the water evaporates. Methyl cellulose can be used to thicken the water surrounding an organism, slowing its movements, or a small amount of alcohol (2%-3%) can be used to narcotize the animals.

If you cannot stop the movement in any convenient way, use the fastest film your camera can handle and brightfield illumination. Try a lower power objective. If the specimen in the photo is now too small, the print can be enlarged, at least to some extent, without introducing empty magnification. The enlarged photo can then be cropped. You may also want to experiment with replacing the illuminator with a much more powerful one, possibly augmented with a remote flash. Place a disk of heat absorbing glass into the light path to protect objectives and specimen.

Video

The simplest approach to video photomicrography is the ready-made package. Several companies sell closed circuit television (CCTV) systems ready to use with their microscopes. Very often these color systems cost more than three times what a microscope alone would cost.

You can piece together a system using an inexpensive, solid state, black and white camera and monitor. This is better if money is scarce and high resolution is needed. A video camera with a removable lens is required. The setup is very much like the setup for an SLR camera, except that a C adapter is screwed to the microscope adapter's T threads, and the camera's internal C threads are screwed to the C adapter. In improved setups a relay lens system screws to the camera's internal C thread, the microscope eyepiece is removed, and the relay lens system inserts into the microscope body tube.

If a monitor display is all that is needed, cable the monitor to the camera, and the project is complete. If a permanent recording is needed, the camera must be cabled to a VCR and the VCR cabled to the monitor. Be sure that the components are compatible with each other.

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Measuring

The most commonly used system for measuring microscopic objects employs an ocular micrometer calibrated to a stage micrometer. The stage micrometer is a slide engraved or printed with a small scale. The ocular micrometer is a diopter adjustable (focusing) eyepiece with a reticle that fits inside. The reticle is also engraved or printed with a small scale or pattern.

First the reticle is brought into focus within the focusing eyepiece. The stage micrometer is centered on the stage and brought into focus with the objective, and the eyepiece is rotated until the two scales overlap. Start counting both scales at a point where markings on the scales visually overlap. Continue counting to a point where the markings overlap again. We know the size of each stage micrometer division and the two counts, so it is easy to work out the size of an individual reticle mark:

Sc -stage micrometer division count
Rc -reticle division count
Sd -stage micrometer division size
Rd -reticle division size
Rd = (Sc * Sd) / Rc.

Here is an example of the calculation. Assume that you counted 12 divisions on the stage micrometer and 27 divisions on the reticle to span these 12 divisions. Assume that the stage micrometer is divided so that each mark represents 20 micrometers. 12 X 20 micrometers is 240 micrometers for the entire span of the stage count. We know that the entire span of the reticle count is the same size as the entire span of the stage micrometer count. Therefore, the size of one reticle division is 240/27 = 8.888 micrometers.

This process must be repeated for each objective, and the reticle division sizes recorded. Thereafter, one simply counts the number of reticle divisions that an object spans, and multiplies this by the size of each reticle division to compute the size of the object.

Reticles come in many patterns. Grids can be used to estimate areas by counting the number of grid squares appearing inside an object. Grids are also useful for particle counts. Concentric circle patterns can be used to estimate the diameter and area of round objects. Crossed micrometers allow quick measurement of an object in two directions. Protractor reticles can measure angles.

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Image Enhancement

The personal computer can be used to manipulate images. There are computer programs, available for most operating systems, that perform image enhancement. The first step in this process is to move the image into the computer.

For photomicrographs, a device called a scanner is needed. The computer may be able to directly read the disks from a disk camera. For video photomicrographs, a video capture board compatible with both the computer and video camera is needed. These boards and scanners usually come with their own image capturing software. The image capturing software supports various standards for writing graphics files to the computer's storage devices. At least one of these file types must be readable by the image enhancement software.

There are many different enhancement techniques that can be used to emphasize different aspects of an image. Indeed, good software can bring out structures that even the most expensive microscopes cannot show clearly. Edges can be detected and enhanced; smoothing removes noise created by glare; and thresholding can be used to highlight parts of an image that are near some brightness value. Black-and-white images can be displayed in pseudocolor, which substitutes a different color for each brightness value range. Color images can be displayed as values of black, white, and gray.

The possibilities are endless, and the field of image enhancement is evolving quickly. Algorithms that learn, known as neural networks, have even been added to some systems. These can be trained to enhance whatever aspects of the image you want to see better. Check computer and camera magazines for new products. You may be surprised to find that image enhancement may be within your budget.

To recapitulate: Search for a video capture board or scanner that is compatible with your computer. Then find enhancement software that is compatible with the files written by the board or scanner and its software. To be sure that everything will work together, send for specifications from several sources before buying.

Some photo finishers are beginning to provide image enhancement facilities, and you may prefer to work with them instead of doing your own.

2006 update: This image enhancement section was written in 1993 and is definitely beginning to look dated. A wonderful free program you should download is the Gimp, available at http://www.gimp.org. You can do lots of interesting things to images with it. Also, the talk about video capture boards is still valid for a few devices, but USB2 port connections are becoming more common and do not require you to open your computer. Scanners are also less often needed because you can load images from commercial digital cameras right into your computer. And today, you can make photographic prints with a hundred dollar printer, and commercial digital cameras make good photomicrographs.

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