Confocal scanning optical microscopy and three‐dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out‐of‐locus light and provides a greater resolution than the wide‐field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction‐limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two‐dimensional image. Thus, confocal scanning optical microscopy allows accurate non‐invasive optical sectioning and further three‐dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three‐dimensional imaging.