Confocal scanning optical microscopy and its applications for biological specimens

Abstract

ABSTRACT Confocal scanning optical microscopy (CSOM) is a new optical microscopic technique, which offers significant advantages over conventional microscopy. In laser scanning optical microscopy (SOM), the specimen is scanned by a diffractionlimited spot of laser light, and light transmitted or reflected by the in-focus illuminated volume element (voxel) of the specimen, or the fluorescence emission excited within it by the incident light, is focused onto a photodetector. As the illuminating spot is scanned over the specimen, the electrical output from this detector is displayed at the appropriate spatial position on a TV monitor, thus building up a two-dimensional image. In the confocal mode, an aperture, usually slightly smaller in diameter than the Airy disc image, is positioned in the image plane in front of the detector, at a position confocal with the in-focus voxel. Light emanating from this in-focus voxel thus passes through the aperture to the detector, while that from any region above or below the focal plane is defocused at the aperture plane and is thus largely prevented from reaching the detector, contributing essentially nothing to the confocal image. It is this ability to reduce out-of-focus blur, and thus permit accurate non-invasive optical sectioning, that makes confocal scanning microscopy so well suited for the imaging and three-dimensional tomography of stained biological specimens. In this review, I explain the principles of scanning optical microscopy and blur-free confocal imaging, discuss the various imaging modes of confocal microscopy, and illustrate some of its early applications.