Confocal Microscopy

Confocal microscopy has become an essential tool for imaging biological samples. In this chapter, we give a brief history of the development of confocal microscopy, followed by a discussion of the theory of confocal microscopy and the effect of the pinhole on the lateral and axial resolution. We also discuss some of the different scanning possibilities currently used in confocal microscopy. After discussing the fundamentals of confocal microscopy, we mention some fluorescence techniques where confocal microscopy plays an essential role. The standard application of confocal microscopy is imaging. However, there are applications of confocal microscopy that do not require imaging. These include fluorescence correlation spectroscopy and single-molecule burst analysis. The image information available from confocal microscopy can also be used with other types of advanced fluorescence methods. As examples, we discuss the number and brightness analysis and raster image correlation spectroscopy.