Laser Scanning Confocal Microscopy

Confocal microscopy images light from a thin confocal slice rather than from the entire specimen. Fluorescence from the specimen is focused by the objective lens through a pinhole aperture to a photomultiplier. Fluorescence from out-of focus planes above and below the specimen plane strikes the walls of the pinhole aperture and is not transmitted. Thus, only fluorescence arising from the in-focus specimen plane is detected by the photomultiplier. High excitation energies provided by lasers (Helium-neon and Argon) lead to equivalent signal intensities from a thin optical slice as from the entire specimen.

The confocal method has several advantages over wide-field fluorescence microscopy. The suppression of out-of-focus objects results in improved image contrast. Therefore, confocal microscopy is particularly well suited to examination of thick specimens where out-of-focus light would obscure details using conventional microscopy.

Another advantage is that it allows the optical sectioning of a three-dimensional object and subsequent three-dimensional visualization using 3-D rendering software. Images found in this atlas were obtained using a Zeiss Laser-Scanning Confocal Microscope with a Sony monitor. The confocal microscope is linked to an IBM RS/6000 Unix workstation where image files were transferred for further processing using Adobe Photoshop.

Comparison of wide-field versus confocal microscopy methods.

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