Confocal microscopy

Abstract

Fluorescence microscopy, whether using immunolabeling or expressible markers such as green fluorescent protein (GFP), has become one of the major tools of the cell biologist. In the fluorescence microscope we can identify cellular structures, organelles and macromolecular assemblies with exquisite precision, but because these structures appear bright on a dark background, strongly labeled objects outside the plane of focus can become extremely distracting. In the worst case they can completely swamp fine details that are in focus. The confocal microscope uses an ingenious bit of optical engineering to overcome this problem, and can in fact create fully three-dimensional images of complex specimens. The principle of confocal optics goes back more than 50 yr, but early implementations were clumsy, and both the mechanical and electronic components then available limited their performance. Most early instruments worked in reflection, not fluorescence, which was useful in material and engineering sciences but had only limited application in biology. Just 20 yr ago, in the late 1980s, the first confocal fluorescence microscopes appeared (1,2) and had an immediate impact in cell biology. Fortunately, too, at that time personal computers (though primitive by modern standards) had advanced to the point where they could do useful things with digital images, even if they were still excruciatingly slow when it came to rendering three-dimensional (3D) data. © 2008 Humana Press.