Confocal Scanning Light Microscopy

Abstract

Confocal scanning light microscopy, a new type of microscopy, has generated considerable excitement. It gives higher resolution and thinner noninvasive optical sections, or planar views, than those obtained by classical bright-field or dark-field microscopy, and increased contrast is another major advantage. Fluorescence microscopy(1) is also greatly improved by using confocal scanning light microscopy, since three-dimensional views can be generated, which lend themselves well to digital image processing and possibly holography. In an image-processing system, a hundred or more very thin optical sections can be stored and combined into a composite, three-dimensional image. Then a display of the total three-dimensional view, or selected parts, can be generated. The composite three-dimensional views can resemble those from scanning electron microscopy, but the specimen does not have to be in a vacuum, as with normal scanning electron microscopy. Currently the technique is used only for reflected light, bright-field and fluorescent microscopies, but additional techniques are under development. Transmission and polarized light microscopes should be available in the future. The confocal microscopical concept was patented in 1957, but the technique has only recently become more common. Delay in its development is probably due to the only recent common application of lasers and scanning techniques. Currently there are some complications with all varieties of these instruments, and efforts are continuing to decrease these.