Confocal microscopy utilizes coherent light emitted by a laser system (excitation source) that passes through a pinhole to exclude out-of-focus background fluorescence from detection. Thus, this technique allows three-dimensional sectioning into thicker tissues. However, the excitation light generates fluorescence, and thus produces photobleaching and phototoxicity throughout the specimen, even though signal is only collected from within the plane of focus. Furthermore, the penetration depth in confocal microscopy is limited by absorption of excitation energy throughout the beam path, and by specimen scattering of both the excitation and emission photons.
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| Rhodamine-phalloidin labeling of actin cytoskeleton in a salivary gland slice | Alpha-actinin (green) and atrial natriuretic peptide (red) labeling in a single heart cell. |