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 μm) slices up to 200 μm below the surface of a transparent tissue: With microscopes running under normal conditions, the optical section thickness will be >1 μm and the effective penetration into enamel and dentin a maximum of 100 μm. 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 μm) 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 ...