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.
Use of a camera on the microscope is straightforward. Merely center the
object to be photographed, focus using the camera viewer, 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) is too
grainy to be used for effective work. In general, the faster the film, the less
inherent resolution it will have. As in all things in photography, a compromise
is called for. |
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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 (for photography
of chromosomes), to moderate (used for general use), and low is not
used in photomicrography. This same film can be used for copy work, since it
reproduces images that are black and white.
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 useful for routine viewing as well as photography.
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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
produce an image that is a medium gray. If you have a spot meter, be sure the
spot is placed over an object that should be gray in the final image. If you have
an averaging meter, be sure there is sufficient material in the viewfinder for 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 so 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.
For 35-mm cameras, be sure to rewind the film when all exposures have
been completed. |
Processing
After exposure of the film, it needs to be processed. Processing of black and
white film has 3 steps. Develop the film, stop it from developing, and fix the
emulsion so that it is no longer light-sensitive. Or, you can send your film out
for processing.
Macrophotography
Macrophotography is used to record things that are 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 also, the type of film used must be changed.
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