Optical Sectioning and Confocal Microscopy

This chapter discusses the stratagems and approaches to imaging that are available to reduce blurring in the final image or are required to collect blur-free 2D and 3D image data sets. It explains how the point-scanning confocal and other optical sectioning microscopes work. The single-beam point-scanning confocal is the most widespread and popular of all the optical sectioning fluorescence microscopes, and several designs are available on the market. The confocal microscope is so-called because the illumination pinhole, the plane of focus within the specimen and the detector pinhole are all situated at conjugate focal planes. The chapter shows the generic features of a typical laser-scanning confocal microscope. Confocal microscopes clearly offer considerable advantages over widefield fluorescence microscopy. Programmable array and line-scanning microscopes are designs that attempt to increase the frame rate, and thus the temporal resolution, of the laser-scanning confocal microscope.