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Interactive Tutorials

Didymium Filters for Color Photomicrography

The intensities and hues generated by most biological tissue stains will reproduce very well on color film, but some stains tend to appear washed out or have their colors shifted, especially in multiple stain mixtures. In many instances, color compensating filters can help restore most or all of the lost color, but too much filtration can affect the color of neighboring stained features and the background. This problem occurs with the popular stains Eosin, Fuchsin, and Methylene Blue, which are not reproduced very well on most color films. Often, tissues stained with these dyes, either singly or in mixtures, appear muddy and lacking in color saturation. Explore, in this interactive tutorial, how didymium filters enhance intensities and hues of specimens stained with a variety of dyes for color photomicrography.

The tutorial initializes with a randomly selected specimen appearing in the window without the advantage of being viewed through a didymium filter. In order to operate the tutorial, toggle the Didymium Filter into and out of the virtual microscope light path with the radio buttons labeled In and Out. This action allows the visitor to view the effects that the filter has on the image of stained specimens. After viewing the didymium filter action, a new specimen can be chosen from the palette using the Choose A Specimen pull-down menu.

To counteract the effects of poor stain rendition, microscopists often employ a didymium filter, which contains a combination of rare earth elements dissolved in glass. Didymium filters remove a portion of the orange region of the visible light spectrum, an effect that increases the color saturation intensity of brown, blue, and red-stained features in the specimen. The extinction coefficient is highest between 580 and 590 nanometers for the didymium filter, allowing the filter to remove most of the orange and yellow colors that tend to dull red and blue tones in stained thin sections.

Didymium filters are produced in very thin glass sections with a thickness of between one and two millimeters, because the amount of light absorbed by the filter is dependent upon filter thickness. Thicker didymium filters can introduce artifacts such as colored backgrounds and degradation of other stain colors. Most major microscope manufacturers offer a didymium filter of proper dimensions as an optional accessory.

In some instances, the background of a biological specimen can become lightly colored during the staining procedure, an undesirable artifact. To avoid this, color compensating filters can be used to subtract the background color, provided it is not too saturated. Another problem with stained thin sections is coloration of the mounting medium. Canada balsam, a popular medium for preparing thin sections, will usually turn light yellow with age or in whole mount specimens. A blue color compensating filter (CC10-20B) will usually overcome this problem, unless the specimen is a very thick whole mount. In this case it may be impossible to completely neutralize the yellow tint.

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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