Photography Through the Microscope

Photomicrography, microphotography, photography through the microscope or whatever you wish to call it, is quite different from normal daylight or flash photography to which most are much more accustomed. Light sources are different, so color balance becomes important with photomicrography. The illumination is reduced compared to daylight or flash, so reciprocity failure of film is an issue. This is especially true in the case of fluorescence photography where the background is nearly black.

Obtaining a good photomicrograph involves setting up microscope illumination to get the best image possible and then taking the photograph with attention to the photographic principles involved.

To take a good micrograph, the microscope illumination path must be set up properly and optimized for the particular specimen to be photographed.

With transmitted light images, this means making sure that Köhler illumination is set up properly. One should consider using a lens of a higher numerical aperture if there is not enough detail in the image and to get the best resolution. The intermediate plane aperture should be stopped down to reduce glare as well as bring out the detail in the image. For color photography, a tungsten or halogen light source should be turned up as bright as possible (3200 K) and a blue conversion filter should be placed in the light to convert the light to that approximating 5500 K. This balances the color and compensates for the yellow of the filament light source. Another alternative is to use a xenon arc lamp which gives white light and provides a full color spectrum. If one is looking at H&E staining (hemotoxylin/eosin), use of a didymium filter (BG20) in the light will enhance the reds. For black and white photography, a very narrow band green filter should be placed in the light path. This will increase the contrast in the final images.

With fluorescence microscopy, one should use the highest numerical aperture possible for the magnification desired, especially if the staining is dim. A higher numerical aperture will pull in more light to the lens.

It is recommended that you bracket your exposures. This means taking two or three exposures at, above, and below the film's rated speed. If the film is ASA 400, this means -1 stop, ASA 800; at 0, ASA 400, and +1 stop, ASA 200. An increase in a stop will double the exposure time. Bracketing your exposures will increase the probability that you will get at least one very good exposure of the particular image. Bracketing is a good idea for two reasons: 1) photomicrography is not as easy as normal daylight or flash photography. Your exposure times are normally a lot longer and reciprocity failure of films must be taken into account (see below). 2) it is a waste of time and energy to come back an rephotograph something for which you got an exposure which was too dark or too light. Your time is much more valuable than a couple of rolls of film. Also, if you are doing fluorescence, it is very probable that your specimens will have faded and not be as good as the first time you photographed them.

Reciprocity Failure

What is reciprocity failure and why is it important to the photomicrographer? Photographic films have a property which is undesirable for microscopists. This is that they tend to lose the ability to absorb light (be exposed) the longer they are exposed to light. This is known as reciprocity failure. It usually occurs with the more commonly available photographic films for exposure times beyond an eighth of a second. Exposures taken with microscopes often extend way beyond this time. Reciprocity failure can in most cases be compensated by increasing the length of the exposure beyond what an exposure meter normally would indicate for a given exposure time. And, of course, reciprocity failure becomes even more important for fluorescence photomicrography because of the long exposures required. Reciprocity failure is a characteristic which varies with the type of film and must either be determined by experimentation or information must be obtained from the film manufacturer. It is desirable for the photomicrographer to use a film with as low a reciprocity failure as possible, especially for fluorescence applications.

How can we compensate for reciprocity failure? First, one can bracket exposures. Second, one can obtain information from the film manufacturer about the reciprocity characteristics of the film in question. Then manual exposures could be taken at several different times and compared with exposure readings taken at the same time. This is somewhat laborious. Some microscope photographic systems have taken reciprocity failure into account in their design. The Zeiss MC 100 photographic module with the Zeiss LSM 410 confocal microscope has a reciprocity setting. Different films are assigned reciprocity numbers based on the characteristics of the film given by the manufacturer. This number can be entered onto a keypad. A list of reciprocity numbers for the commonly used film types in the Institute is posted behind the microscope for your use. When one takes fluorescence micrographs of weakly stained specimens, exposure readings might become so long as to make the latter part of such an exposure meaningless due to reciprocity failure. Exposing for longer than two (or at the very longest, three) minutes tends to only increase the background brightness of a fluorescence image. In this case, it is better to set a base exposure ( a 0 setting) at something between 20 and 40 seconds and bracket exposures one above and one below. For microscopes which only have a camera mounted to the microscope, it is best to do bracketed manual timed exposures for fluorescence microscopy. Light levels are too low for normal daylight exposure meters in those cameras to get a useful low light exposure reading. For microscope photographic systems which have no reciprocity failure conpensation, it is probably best to just to use a film with low reciprocity failure and to bracket exposures. 

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