Photomicrography, microphotography, photography through the microscope is different from normal daylight or flash photography to which most are accustomed. Light sources are different, so color balance becomes important with photomicrography. The illumination is less 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.
Reciprocity failure – the undesirable property of photographic films they tend to lose the ability to absorb light (be exposed) the longer they are exposed to lid. 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.
There are ways to resolve reciprocity failure. Bracket the exposures. 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 2) it is a waste of time and energy to come back and rephotograph something for which you got an exposure which was too dark or too light 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. Some microscope photographic systems have taken reciprocity failure into account in their design. They provide reciprocity setting. Different films are assigned reciprocity numbers based on the characteristics of the film given by the manufacturer. A list of reciprocity numbers for the commonly used film types in the Institute is posted behind the microscope for your use. 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 compensation, it is best to just to use a film with low reciprocity failure and to bracket exposures.
Photography through the Microscope To obtain a good photomicrograph, microscope illumination must be set up to get the best image possible and then taking the photograph with attention to the photographic principles involved. The microscope illumination path must be set up properly and optimized for the particular specimen to be photographed.
In fluorescence microscopy, the highest possible numerical aperture should be use for the magnification desired, especially if the staining is dim. A higher numerical aperture will pull in more light to the lens.
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 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.
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