A computer-assisted video technique for preparing high resolution pictures and stereograms from thick specimens.

A computer-assisted video technique is presented for rapidly and accurately gathering, storing and depicting three-dimensional anatomical structures in thick specimens. Several optical sections through the specimen are combined to produce high-resolution photographs with essentially infinite depth-of-field. Further, the depth information implicit in the series of optical sections makes the creation of stereoscopic pairs relatively simple. The technique employs a real-time digitizing frame store and a computer. A video camera is attached to a microscope and successive optical sections are stored digitally as the plane of focus is systematically changed. After storage, the image of each optical section is enhanced to emphasize elements that are sharply focussed. The final two-dimensional image is generated by selecting for each point in the final picture the darkest grey value occurring at the corresponding point in any of the pictures in the through-focus series. A picture with essentially infinite depth-of-field is produced when points of correspondence in the series are determined by a ray passing normal to the plane of optical section. Right and left pictures for a stereoscopic pair are produced when points of correspondence are determined by a ray slanting either left or right of normal. This technique is illustrated with cobalt chloride-filled neurons from whole-mounted cricket ganglia, with HRP-filled axons from whole-mounted goldfish tectum, with Golgi-Kopsch-impregnated neurons from cat visual cortex, and with sections of cobalt chloride-filled antennal afferents in cricket.