Fact and artefact in confocal microscopy.

High-resolution confocal microscopic images may be made of either the surface of a sample or beneath the surface. These images can be likened to optical tomograms, giving thin (> 0.35 micron) slices up to 200 microns below the surface of a transparent tissue: With microscopes running under normal conditions, the optical section thickness will be > 1 micron and the effective penetration into enamel and dentin a maximum of 100 microns. For maximum resolution, high-quality, high-numerical-aperture objectives should be used. Refractive index matching of the lens immersion media and the substrate will avoid distortions of images in the optical axis. Such errors could occur when imaging a considerable distance (> 40 microns) into a cell containing water, with an oil immersion objective above the cover slip. Care should be taken in the interpretation of computerized z axis reconstructions made from serial optical sections: Their validity should be checked with equivalent views made with the sample oriented in the same direction as the reconstruction. The use of fluorescent dyes in microscopy is a very powerful investigative technique. It is important that the dyes used not be labile and that they be well-fixed to the materials being examined, or the images may indicate the dye distribution rather than the material to which it is "attached". Multiple labeling experiments must have crossover control experiments to verify the distribution of the individual dyes. Valuable information can often be gained by combining information from both reflection and fluorescence images. Two-photon laser excitation of dyes gives the potential for greater depth penetration and improved resolution.