AUTOMATED 3-DIMENSIONAL CHARACTERIZATION OF OSTEOCLASTIC RESORPTION LACUNAE BY STEREOSCOPIC SCANNING ELECTRON-MICROSCOPY

The use of stereoscopic scanning electron microscopy to analyze quanti tatively the topography of excavations made by osteoclasts in slices o f devitalized cortical bone was evaluated. Using this innovative techn ique, the need mechanically to tilt the specimen stage to gather three -dimensional information is obviated by instead tilting the electron b eam both to produce real-time stereo pairs and to gather measurement d ata. 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 corr ect the height of the tilt axis and lens focus in a feedback loop, gen erating a quantitative measurement of height difference. With this app roach, relative heights of individual points, line profiles, area topo graphy maps, and three-dimensional reconstructions of excavations were obtained rapidly and at high resolution. This approach combines the a dvantage in topographic data acquisition of confocal microscopy with t he advantages of the increased resolution and focal depth of scanning electron microscopy (SEM). The technique should facilitate not only th e topographic analysis of osteoclastic excavations in bone slices at h igh resolution but also the three-dimensional analysis of the structur e of bone tissue.