Education in Microscopy and Digital Imaging

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.

Click here to access the complete article.