Three-dimensional specimen reconstruction by confocal microscopy and digital image processing

Abstract

In a confocal microscope point-wise illumination of the specimen is used, often in the form of a focused laser beam that is scanned in a raster pattern. The reflected or fluorescent light from the specimen is focused onto a small aperture in front of the detector. This imaging method has advantages such as improved resolution and less stray light. In addition optical sectioning is possible, whereby thick specimens can be studied layer by layer without the need for mechanical sectioning. By combining information from a large number of optical sections, registered at different depths, a three-dimensional reconstruction of the specimen can be made. Digital image processing can be used both for producing images for visual display and for extracting quantitative information. Quantitative evaluation is hampered in many cases by factors such as absorption, scattering, and refraction of the light travelling through the specimen volume.