Cameras used for photomicrography generally consist of either a standard "single-lens-reflex" (SLR) 35-mm camera body mounted via a tube to the microscope (which uses its own integral exposure meter) or consist of a specially designed unit that is integrated into a dedicated "photomicroscope" (which will generally have more sophisticated exposure controls and which may use either 35-mm or larger format film). Although film is still widely used, digital image acquisition is increasingly common. However, regardless of the type of camera, the central principle of photomicrography is always to optimize the image (in terms of contrast, resolution and color) at the point of collection, be it on a photographic negative, CCD camera, video camera, or PMT. This must be achieved by having the correct focus and lighting conditions for optimal contrast and color faithfulness.
Since microscope cameras have a fixed aperture, exposure times depend only on lighting levels and the sensitivity of the digital collection device or photographic media. Long exposure times, particularly in fluorescence photomicrography, are usual. For digital cameras, long exposure times result in problems related to thermal noise. Consequently, images are usually averaged (see Subheading 3.7.1.), and highly sensitive CCD chips are used at low temperatures. For film, extended exposures (beyond as little as perhaps 2 s) result in particular problems for color photomicrography relating to reciprocity failure (see Subheading 3.9.1.).
Light from the specimen must be directed to the camera and focused onto the surface of the film. This usually involves redirecting or splitting the light path from the eyepieces to the camera tube and ensuring that what is in focus for the eyepiece is also in focus on the camera. Such parfocality cannot always be assumed for a microscope with a 35-mm camera attachment via a camera tube, and it is advisable to check that the image is correctly focused for the camera through the eyepiece of the SLR camera body. Photomicroscopes are designed so that camera and eyepieces are parfocal when the diopter of each eyepiece is correctly adjusted for the observer (see Note 3). This is usually achieved by focusing the crosshairs of a reticule placed or projected onto the eyepiece diaphragm. The diopter ring is adjusted such that the reticule image is sharp when the eyes of the observer are relaxed. The simplest way to relax eye muscles is to glance briefly at a distant object before focusing the crosshairs. When the specimen is focused, crosshairs should also remain sharply in focus. Particular care must be taken with objectives of a low magnification and especially with dissection microscopes, where the increased depth of field allows the observer's eyes to accommodate across a greater depth within the specimen. In such situations, a "focus telescope" may be useful to focus accurately on a specific optical plane.
Black and white is still widely used for publication of photomicrographs and offers certain advantages over color photography. Monochrome films are less sensitive to variations in exposure times and, in particular, are unlikely to suffer reciprocity law failure where exposure times are under 1 min (see Subheading 3.9.1.). Films vary in their speed (measured by ASA or DIN) reflecting their sensitivity to light: generally, faster film speeds result in a coarser-grain image. Films may also vary in their gradation or contrast, which can be characterized by the "y" of their emulsion. Within a reasonable exposure time, the response of an emulsion to increasing exposure is linear. Gamma (Y) is defined as the tan of the angle of the linear portion of this response curve (a) and is a useful guide to the contrast of the film. If y > 1 (a > 45°), then the emulsion has an enhanced reaction to changes in intensity and, therefore, a "hard" contrast. If Y < 1 (a < 45°), then the film has a "soft" contrast. Emulsion may also react slightly differently to different colors of light. A panchromatic film will have an approximately even sensitivity across all wavelengths, but may still be less responsive to deep-reds. A selection of films used in black-and-white photomicrography is as follows.
T-MAX 100 ASA (soft contrast) ILFORD FP4 (intermediate contrast) KODAK TECHNICAL PAN 125 ASA (high contrast) T-MAX 400 ASA (for fluorescence)
AGFA SCALA 200 ASA (reversal film for B&W transparencies) 18.104.22.168. Filters
Black-and-white photomicrography allows the use of color filters to optimize the contrast of objects within the specimen. Color filtering can be used to convert differences in color contrast into differences in perceived intensity. An absorption filter of one color will remove wavelengths of the complementary color deepening the contrast between such objects and the background. For example, a blue-green or cyan filter will darken the reddish-brown (DAB) product used in histological peroxidase reactions. The effects of different colors of contrast filters are shown in the following table (adapted from ref. 1).
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