Appendix I: Photography


The use of photography within a cell biology laboratory allows for the capture of data and images for processing at a later time. It also is an excellent means of preparing materials for presentation, either through projection slides, or through illustrations.

PHOTOMICROGRAPHY

Photographically recording visual images observed through a light microscope is a useful means of obtaining a permanent record of activities. Using photomicrographs is the main means of recording electron microscope images.

Photomicrography begins with proper microscope use. It is important that the microscope be in good operation, clean and centered. Ideally, the microscope will be equipped with either a trinocular head, or a built in camera port. Excellent results can be had, however, by attaching a 35 mm single lens reflex camera to an eyepiece by way of an inexpensive adapter and a tripod or copy stand. The adapters are designed to fit in place of the lens, and the tripod is to take the weight of the camera off of the lens tube. The reflex camera is useful since it has its own focusing screen. Although you will not get a sharp image in the camera (because of the ground glass image plate), the negative will be sharp. Special camera attachments are available from the microscope manufacturers which incorporate excellent optical focusing devices, but these are costly. If a lot of photomicrography is to be performed, however, they are well worth the cost.

Use of a camera on the microscope is straightforward. Merely center the object to be photographed, focus using the camera viewer (that is, do not focus using the microscope eye piece) and depress the camera shutter button. Equipping the shutter with a shutter release cable will help prevent vibrations. This assumes that you have the proper exposure.

Exposure and film type are the major problems of photomicrography. For most microscopes using a tungsten lamp source, there is very little light reaching the camera. Film that has a high enough exposure index (ASA speed) are too grainy to be used for effective work. In general, the faster the film the less inherent resolution the film will have. As in all things in photography, a compromise is called for.

Moreover, most film sold for general use has a thicker film emulsion than is desirable. The microscope projects an image of very low contrast, with low light intensity. A thick emulsion tends to lower the contrast even more. This results in photographs that are all gray, with no highlights (black and white). The tonal range is reduced significantly using general film for photomicrography.

Use Kodak Technical Pan Film at an ASA of 100 for photomicrography. This is a thin emulsion film with extremely high contrast. The contrast can even be controlled through the developing process and ranges from High (used for photographing chromosomes), to moderate (used for general use) and low (not used in photomicrography). This same film can be used for copy work, since it reproduces images which are black and white. In general, you can not have too much contrast in a photomicrograph, but it is possible with this film.

Another means of increasing contrast is the use of colored filters within the microscope light path. Use a contrasting color to the object you wish to photograph. For example, chromosomes stained with aceto-orcein (dark red) can be contrast enhanced by the use of a green filter. Human chromosome spreads stained with Giemsa (blue) can be enhanced by the use of a red filter. This trick is also useful for routine viewing as well as photography.

The use of filters will increase the necessary exposure time. Technical Pan Film is also a relatively slow film. To establish the proper exposure, use the light meter built into the camera. If no light meter is available, you will have to shoot a roll of film and bracket several exposures to determine which is best. When using the built in meter, remember that all light meters are designed to give an image which is a medium gray. If you have a spot meter, be sure the spot is placed over an object which should be gray in the final image. If you have an averaging meter, be sure there is sufficient material in the viewfinder to give a proper average exposure. If you do not know whether you have a spot or averaging meter, find out. This is not trivial. Suppose you wish to photograph a chromosome spread. The chromosomes are typically less than 1-2% of the field of view. The meter will adjust the exposure such that the white field of light is exposed as gray, and your chromosomes will appear as darker gray on a gray field - in other words, extremely murky looking. Performing karyotype analysis on this type of image is difficult or impossible.

Bracket all exposures. Once you have determined the appropriate exposure, be sure to take several photographs. If, for example, the meter says 1/60 sec exposure, take another at 1/30 and one at 1/100. This process is known as bracketing the exposure to ensure that one is correct. Kodak Technical Pan Film is somewhat forgiving for poor exposure, but only somewhat. For black and white film, if you err on exposure, overexpose. This is the exact opposite for color positive film (slides).

For 35 mm cameras, be sure to rewind the film when all exposures have been completed.

PROCESSING

After exposure of the film, the film needs to be processed. Processing of black and white film has three steps. Develop the film, Stop it from developing, and Fix the emulsion so that it is no longer light sensitive.

You can send your film out for processing, but it will take longer, cost more, and in general you will be more pleased with your own work. The procedure requires about 30-40 minutes and can be done while cleaning up the lab.

PERFORM IN TOTAL DARKNESS:

Remove the film from the its cannister and roll it onto the developing tank reel. Place the film and reel into the developing tank and place the light prrof lid onto the tank. You may then turn on the lights. 1

For processing, follow the following steps:

  1. Select the proper developer based on the film manufacter's recommendations. 2 Dilute the developer as recommended and measure the temperature.
    For Technical Pan film exposed at 100 ASA, use Kodak D-19 developer dilution D at a temperature of 21 ° C.

  2. Pour the diluted developer into the tank and allow the solution to remain for the recommended time of development ( 6 20minutes for TP film). The development time is time and temperature dependent. Be sure to use the correct combination for the film used. During the development stage, gently swirl the solution in the tank once every 30 seconds. It also helps to invert the tank during this stage if the tank is equipped with a lid. If inverted, give the tank a mild rap on the bench top as you set it down. This will dislodge any trapped air bubbles from the film reel. Do not slam the tank down, it takes only a mild tap!

  3. Stop the developing by pouring the developer from the tank and replacing it with Kodak Indicator Stop for 30 seconds.

  4. Pour off the stop bath and replace it with Kodak Fixer for a period of 6-8 minutes. Kodak Rapid Fix may be substituted for a period of 2 minutes.

  5. Pour off the fixer and replace with a 1:4 dilution of Kodak Hypo Clearing Agent for 2 minutes.

  6. Pour off the clearing agent, open the tank and wash the film in running water for a period of no less than 5 minutes. After washing, rinse the film in distilled water (you may use Photoflo if available) and air dry the film.

PRINTING

Processing a roll of film results in a strip of negatives. For much of the photographic work in cell biology, it is not necessary to do anything with these negatives, except to store them, or use them directly for observation. Holding the negatives up to a light source, and viewing with a hand held magnifying lens is often sufficient. However, for presentation work, or for karyotype analysis, where you wish to cut out the chromosomes and rearrange them in some manner, a print needs to be made.

Printing the image is a more time consuming process than developing the film, requires a darkroom and is more expensive. As a minimum, there is a need for a photographic enlarger, developing trays and a good timer. Photographic paper is more expensive than film.

The basic process involves inserting the negative into the carrier of an enlarger, exposing a sheet of photographic paper to the projected negative and then processing the paper film in a manner similar to that for the film. The exception is that the Developer is switched to Dektol and the film is processed through trays rather than on a reel and within a tank. There are, however, many variations.

If many prints are to be made, a stabilization processor can be used. This works by virtue of the fact that the paper comes with the developer incorporated into the emulsion. It is activated by passing the paper through a bath of strong alkali, and halted by passing it through a strong acid. This is done automatically by a machine that resembles an old thermofax machine. The paper is fed into the front and rolls out as a photograph from the back. It is stable for about 6 months, unless fixed with a bath of Kodak Fixer, which will make it as permanent as any photograph. Electron microscopy labs will often have one of these instruments available.

For details on printing (which has many aspects of an art form), refer to a text on photographic processing.

MACROPHOTOGRAPHY

Macrophotography is used to record things too large to be viewed in the microscope. This is an excellent means of making permanent records of electrophoresis gels, bands observed during ultracentrifugation, and whatever else you wish to capture on film.

Two changes are required from the use of photography through a microscope. The camera must be removed from the microscope and equipped with a lens, and secondly, the type of film used must be changed.

Very briefly, there are two means of adapting a 35 mm camera for macrophotography. The simplest is to purchase a macro lens. It is preferrable to use a real macro lens rather than a zoom with macro capability. Zoom lenses do not have the inherent resolution suitable for macro work. True macro lenses are, on the other hand, relatively expensive (~$300). An alternative solution is to purchase a lens reversal ring or a set of extension tubes for your camera. These are both inexpensive options. The former, use of a reversal ring, is an excellent means of optaining macro capability by simply turning around the normal 50 mm lens (cost about $10). The use of extension rings allows some variation in the magnification capabilities (~$30).

For occasional photos of electrophoresis gels, it is probably worth investing in a camera specifically designed for that purpose. Fotodyne markets a Polaroid camera gun for this purpose (~$600). At the other extreme is the use of an MP4 Polaroid copy stand ($2,000) for macrophotography. The most economical means of performing routine photography is to purchase a used 1960's Pentax Spotmatic (or equivalent) with lens reversal ring and a microscope adapter. Combined with a bulk film loader and a changing bag, nearly all the required aspects of film recording of data can be readily accomplished at bargain prices.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu