Overview of Fluorescence Analysis with the Confocal Microscope

When fluorescently labeled biological specimens are viewed with a conventional wide-field microscope, a haze of out-of-focus fluorescence is usually created by the overlapping structures within the sample. As we focus through the specimen, our brains have a remarkable ability to discern substantial structural detail. However, the resolution of the images we record on film is degraded by the out-of-focus fluorescence. The confocal microscope can reject out-of-focus information and enhance the contrast of an image because the illumination and the detection are confined to an identical (small) region of the specimen. An overview of the basic principles of a confocal microscope is presented in Fig. 1 and out-lined below.