Automated three-dimensional characterization of osteoclastic resorption lacunae by stereoscopic scanning electron microscopy.

The use of stereoscopic scanning electron microscopy to analyze quantitatively the topography of excavations made by osteoclasts in slices of devitalized cortical bone was evaluated. Using this innovative technique, the need mechanically to tilt the specimen stage to gather three-dimensional information is obviated by instead tilting the electron beam both to produce real-time stereo pairs and to gather measurement data. Based on the comparison of two images of microscopic areas, cross-correlation is used to determine the image shift between the features in the stereo pair. This information is then used dynamically to correct the height of the tilt axis and lens focus in a feedback loop, generating a quantitative measurement of height difference. With this approach, relative heights of individual points, line profiles, area topography maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the advantage in topographic data acquisition of confocal microscopy with the advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only the topographic analysis of osteoclastic excavations in bone slices at high resolution but also the three-dimensional analysis of the structure of bone tissue.