Confocal microscopy: principles and practice

INTRODUCTIONThe confocal microscope is an invaluable tool for high-resolution fluorescence microscopy, and few biological research centers are without at least one such instrument. Much of the interest in confocal microscopy stems from the reliance on fluorescent probes in contemporary biology. Fluorescent-labeled antibodies and ligands are essential tools for localizing specific molecules. Intracellular or membrane-bound fluorescent dyes are widely used to follow morphological changes in cells, and retrograde-transported fluorescent markers have been used to identify living neurons with particular projections for subsequent electrophysiologic or structural study. Voltage- and ion-sensitive indicator dyes have been used to observe patterns of electrical activity in large networks of neurons and in structures too small to be monitored with classic electrode techniques. Green fluorescent protein (GFP) permits the visualization of specific proteins, including engineered functional probes, in essentially unperturbed living tissue. This article outlines the basic principles of confocal microscopy, relevant practical considerations, and various approaches to its implementation, principally from the perspective of visualizing rapid, small-scale phenomena in living tissue.