In transmitted light microscopy, the specimen quality does not always lend itself to easy observation and image recording with excellent contrast in simple brightfield imaging mode. Investigations dealing with inherently low-contrast specimens, such as unstained bacteria, thin tissue slices, and adherent live cells, rely on specialized contrast-enhancing techniques to assist with imaging these virtually transparent samples. In the course of examining unstained specimens, poor light absorption by the specimen results in extremely small variations in the intensity distribution difference between the specimen and the background. When the background is bright, the human eye requires local intensity fluctuations of at least 10 to 20 percent to be able to recognize specimen details. Unfortunately, this level of modulation is seldom seen with transparent specimens, which are usually rendered almost invisible against a background of similar intensity. The term transmitted light, when used in optical microscopy, refers to any imaging modality where light is passed from the illumination source on the opposite side of the specimen to the objective (thus, illumination is transmitted through the specimen). The contrast-enhancing techniques described in this section represent a variety of methods in sample preparation as well as optical tricks that generate intensity changes which are useful for observation and imaging.

Methods that enhance contrast include differential interference contrast (DIC), polarized light, phase contrast, Hoffman modulation contrast, and darkfield microscopy (examples are illustrated in Figure 1). Several of these techniques are limited by light originating in regions removed from the focal plane when imaging thicker plant and animal tissues, while polarized light requires birefringence (usually not present to a significant degree in animal cells) to generate contrast.
Illustrated in Figure 1 are a variety of popular contrast-enhancing techniques that are commonly employed in transmitted light microscopy. The thin tissue section in Figure 1(a) reveals a human basal cell carcinoma stained with eosin and hematoxylin to generate color contrast in brightfield imaging mode. Figure 1(b) shows living HeLa cells in a plastic tissue culture vessel imaged with phase contrast. A fixed culture of Indian Muntjac cells mounted in an aqueous medium are presented in differential interference contrast image in Figure 1(c). A fresh tissue section of mouse heart muscle bathed in aqueous saline solution is displayed in the Figure 1(d) panel, where contrast is generated using Hoffman modulation (or ZEISS VAREL) contrast, an oblique illumination technique. The brilliant bright-on-dark contrast observed with darkfield illumination is shown in Figure 1(e) using an Obelia hydroid specimen. Finally, rabbit skeletal muscle fibers (Figure 1(f)) are among the biological specimens that are birefringent and demonstrate contrast in polarized light.