Nomarski differential‐interference contrast microscopy in transillumination: Its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell‐culture monolayers

Abstract

SUMMARYUnstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright‐field, positive phase contrast, and Nomarski differential‐interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright‐field image); however, Optical sectioning' of darkly stained objects is not possible.Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm.Acridine orange (AO) stained cell‐culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi‐fluorescence or fluorescence in transillumination, and DIC‐fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information.Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.