An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.

TLDR

Scanning confocal microscopes provide superior rejection of out‑of‑focus noise and higher resolution than conventional imaging, and are especially suited to epifluorescence microscopy where a single confocal lens replaces the separate imaging and condenser lenses. A beam‑scanning confocal system that moves the light beam rather than the stage proved faster, easier to use, and produced markedly clearer epifluorescence images, enabling optical sectioning of thick specimens with minimal quality loss.

Abstract

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.