Factors affecting visibility of a target tissue in histologic sections

The objective of histologic techniques is to stain the subject with high specificity and high visibility. Visibility depends on the microscope's resolution and contrast and on the microscopist's skill at optimizing the microscope's image. It also depends on histotechnological factors, which include specificity and differentiation of the stain, density of background staining (particularly in silver stains), innate color, and grayscale contrasts of the dyes in the stains and color and density of the counterstain. If contrast is not optimal, the image should be evaluated on the basis of 2 types of contrast-color and grayscale. Complementary colors have maximum color contrast, and the color triangle is useful in the selection of a suitable counterstain. Grayscale contrast is a function of the density of a stain. If dyes capable of staining the target and backgrounds tissue do not have optimal color contrast, the only method of increasing contrast is to change the grayscale value of one of the stains, usually the counterstain. Colors can have a subconscious effect on a viewer. Depending on whether they are aesthetically pleasing, they may influence the rigor of and time spent on the histopathologic examination. Maximizing the specificity of stains such as hematoxylin, eosin, trichrome, and Luxol fast blue (LFB) depends on optimal differentiation. In differentiation of counterstains such as methylene blue in the Ziehl-Neelsen stain, its recommended density is conveniently expressed as a grayscale value. Independent evaluation of color and grayscale contrasts is very helpful in determining the cause of low contrast in an image. This review discusses aspects of the histotechnique affecting the visibility of tissue components.