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Interactive Tutorials
Filter Control of Image Contrast in Black & White Photomicrography
As a general rule when employing color filters in black & white photomicrography, utilize filters that are complementary to specimen stain color (they absorb most of the predominant wavelengths transmitted by the stain) to maximize the amount of contrast in final images. To achieve a medium level of contrast, use filters that only partially absorb colors displayed by features of interest. Finally, to reduce contrast to a minimum, use filters that have colors identical to those of the specimen. A combination of filters can be used to enhance detail contrast in specimens stained with more than one color. This tutorial explores the use of Kodak Wratten color filters for contrast control in black & white photomicrography when using stained specimens.
To enhance contrast, color filters are added to the microscope light path to absorb stained specimen color, rendering it either a darker or lighter shade of gray. Contrast can be adjusted in this manner by selectively choosing filters that absorb varying amounts of stain color. The images presented in this tutorial can be used to explore these effects. Use the Choose a Specimen pull-down menu to select a stained specimen from the list. After selecting a specimen, use the radio buttons to simulate how black & white photomicrographs will appear when using one of three Kodak Wratten color filters: 25 (red), 58 (green), and 47B (blue). When a filter radio button is selected, the image changes to a black and white photomicrograph showing the results of the filter selection. Use the three filter algorithms, either individually or in groups, to view how filter combinations can use used to adjust contrast in black & white photomicrography.
Most biological specimens lack sufficient color and contrast to be readily imaged in the optical microscope using brightfield illumination. These specimens do not usually absorb visible light to any great extent (they are not good amplitude specimens) and can be seen as a rough outline with some internal detail only when the condenser aperture size is reduced, often to the point of introducing optical artifacts. To circumvent this problem, microscopists often treat biological cells and tissues with reactive organic dyes that will selectively stain and color various portions of the biological architecture. Because the background often appears white or very light gray in brightfield microscopy, the stained tissue will appear colored and superimposed over a light background. This is often sufficient to render the details of interest visible and with enough contrast to provide good photomicrographs.
In many instances, two or more stains of different color are combined to help differentiate between cellular elements that lie close together, producing a color contrast that separates one element from another. For example, cell nuclei can be stained with Hematoxylin, a natural leuco compound extracted from chipped logwood, which selectively binds to chromatin. To provide contrast, Hematoxylin is often combined with Eosin, a red fluorescein dye that stains a variety of cytoplasmic structures. The combination of these two stains renders cells with nuclei stained a deep blue and having cytoplasmic components that are stained red.
Procedures for staining biological tissue range from very simple mixtures of single cells with dye, to complex multi-step tissue sectioning processes that consume a considerable amount of time and materials. Smears can often be stained by simply dropping a mixture of stain and fixative into a culture tube containing the cells to be examined. Tissue sections are more difficult, and require several fixative and sectioning steps prior to being placed onto microscope slides and run through a gamut of stain and wash solutions.
Contributing Authors
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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