Introduction to Confocal Microscopy

Confocal microscopy is a technique in optical imaging that uses point illumination via a spatial pinhole to eliminate outof-focus signals. The excitation light in confocal microscopy is usually provided by a laser to generate high intensities of fluorescence or reflectance from the focal spot. Fluorescence confocal microscopy is the most used in dermatology to analyze ex vivo and in vitro samples. Reflectance confocal microscopy can be used for real-time microscopy and uses melanin as a natural contrast agent. Confocal microscopy has many advantages, including increasing the optical resolution and contrast of an image of a specimen; facilitating reconstruction of 3-D images; enabling collection of serial optical sections from thick specimens; and enabling in vivo imaging without the artifact induced by tissue processing (Pawley, 2006). In addition to LSCM, 3-D images of nonliving samples can also be acquired by SCEM, where an electron beam is used for illumination, resulting in higher resolution compared with onfocal microscopy. Limitations of confocal microscopy include the depth of imaging within thick samples and cost compared with conventional microscopes. The problems of fluorescent probe photobleaching and phototoxicity inherent in conventional fluorescence microscopy are also present with confocal microscopy. Multiphoton microscopy is an alternative strategy for fluorescence microscopy, which offers higher resolution, somewhat greater depth of imaging, and minimal photobleaching. Technologies for microscopy are promising and are still being improved. © 2012 The Society for Investigative Dermatology.