Troubleshooting Errors and Faults
Photomicrography, like any form of photography, is prone to a variety of faults and errors regardless of the sophistication of the microscope equipment or the experience level and skill of the photomicrographer. Errors must be carefully examined to identify the source, which is usually due to either equipment failure, poor specimen preparation, improper technique, or processing mistakes. Use the links below to navigate to our specific discussions on the nature of photomicrography errors and suggested remedies.
Twenty Five Most Common Photomicrography Errors - In search of a quick answer to your photomicrography problem? We discuss the most common errors encountered in everyday photomicrography and provide suggestions for easy solutions.
Microscope Configuration and Other Common Errors - Errors in the configuration of a microscope include incorrect settings of the field and condenser aperture diaphragms, improper illumination adjustment, and optical aberration. Focus and vibration artifacts are also a common source of errors in photomicrography. These configurational errors and related problems are addressed in this section.
Exposure and Color Balance Errors - Exposure and color balance errors are perhaps the most common problems that occur with color photomicrography and have been encountered by all microscopists, both amateur and professional alike. This discussion addresses common problems with color temperature balance adjustments, reciprocity failure, incorrect exposure, and how to correct minor color errors with compensating filters.
Fluorescence Photomicrography - Photomicrography under fluorescence illumination conditions presents a unique set of circumstances posing special problems for the microscopist. Exposure times are often exceedingly long, the specimen's fluorescence may fade during exposure, and totally black backgrounds often inadvertently signal light meters to suggest overexposure.
Microscope Configuration and Photomicrography Errors - We review the most common microscope configuration errors and problems that occur in fluorescence microscopy, and also review several common errors in photomicrography. Use this section as a quick guide to solving elementary problems in fluorescence microscopy and photomicrography.
Color Transparency Errors with Fluorescence Photomicrography - Determination of the correct exposure in photomicrography is much easier when transparency films are being judged. These films have a very narrow exposure latitude, usually one-half to one f-stop, and incorrect exposure is very obvious. With transparency film, a good exposure will reveal specimen details in all areas of the photomicrograph, unless this detail is actually missing in the specimen.
Fluorescence and Differential Interference Contrast Combination Microscopy - Fluorescence microscopy can be combined with contrast enhancing techniques such as differential interference contrast (DIC) illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC then, without relocating the specimen, switching the microscope to fluorescence mode.
Fluorescence and Phase Contrast Combination Microscopy - To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.
Color Transparency (Chrome) Film Errors - Color transparency (or reversal) film currently provides the highest resolution format available for color photomicrography, but this film has very close exposure and development tolerances that can easily lead to errors. In this section, we address common problems and provide illustrations of photomicrograph errors that occur when using color transparency film using a stained thin section of human taste buds as the example specimen. The same errors are also reviewed utilizing a quadruple-stained thin section of alfalfa crown wart fungus to further illustrate common mistakes made with color transparency films.
Errors in Color Transparency Film Encountered with Reflected Light Microscopy - Many of the errors committed by microscopists with reflected light illumination are similar to those observed with transmitted light. This section examines common errors that occur with reflected light photomicrography on color transparency films. Examples of these errors are illustrated by employing the regular array of elements found on the surface of an integrated circuit, a popular specimen choice for reflected light microscopy.
Color Negative Film Errors - Color negative film has a much wider exposure latitude than does transparency (slide) film, with the added bonus that color balance problems and undesirable color casts and shifts can often be corrected during printing. This section reviews common errors encountered with color negative film in photomicrography using a multiple-stained thin section of pine tree tissue infected with blister rust fungus.
Black & White Film Errors - Although used far less often today than in the past, black & white film is still a very important medium for photomicrography, especially with stained specimens in brightfield illumination. Contrast enhancing filters are often employed by photomicrographers to increase detail and clarity in black and white images of stained specimens. Photographic negatives of a multiple-stained thin section of pine tissue are presented in this section to illustrate common errors that occur with black & white photomicrography.
Film Processing Errors - Analysis of faults and errors in photomicrography must include careful differentiation between mistakes in microscopy and those caused by problems with film processing. The links below lead to discussions with illustrated examples of the common errors encountered with processing black & white and color films used in photomicrography.
Color Transparency Film - Processing color transparency film requires considerably more effort and attention than does black & white film. Although the basic steps are similar in nature, color processes require more steps and greater temperature control than do black & white processes. The stability of chemical solutions is also a factor in color processing, because sensitive combinations must be mixed immediately prior to use to avoid rapid deterioration of the components.
Color Negative Film - Evaluation of color negatives is somewhat difficult and requires more skill and experience than do black & white negatives or color transparencies. Highlight areas should not be excessively dense and shadowed features should be sufficiently detailed to render an overall balanced photomicrograph. Judging specific colors in color negatives is complicated by the presence of a mask that produces an orange tint over both the sprocket holes and the image area, including the color dye layers.
Black & White Film - Processing black & white film is a relatively minor task that can easily be accomplished in any laboratory that has a darkroom or access to a closet with subdued lighting. Although a wide spectrum of processing errors are possible with black & white film, the majority are easily identified and corrected. This discussion is intended to help the microscopist distinguish between mistakes that occur with the microscope and those that are the result of faulty film processing.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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