Measurement of osteoclastic resorption pits with a tandem scanning microscope

The functional capacity of isolated osteoclasts is measured by determining the volume of bone or dentine which they can resorb in vitro when cultured on flat tissue slices. Previous SEM stereophotogrammetric evaluations showed that the measurement of total resorptive area per sample, number of pits per sample, area per pit, etc., may be insufficient and that the volume to area ratio may be of utmost importance. A new procedure for the 3-D description and measurement of resorption pits using confocal microscopy has been established. Osteoclasts were cultured on slices of dentine which were prepared as for SEM. They were imaged with a Tracor Northern TSM equipped with piezo-electric translators for the objective lens focusing motion, interfaced to a TN8500 computer, using a TV camera for image transfer. The procedure consisted of generating a zeta map image of the focus depth at which maximum brightness was found at each pixel. Constructing binary masks corresponding to (i) the edge of the resorption pit to define the area beneath which the volume is to be calculated, and (ii) a uniform width at a small distance outside the edge of the pit, enabled the average height of the original surface to be measured and the slice above the pit to be subtracted. After subtraction of the volume element which lay above the top of the original specimen surface, the zeta map is a measure of both the area and volume of the pit.