Reproducible methods for calibrating the backscattered electron signal for quantitative assessment of mineral content in bone.

Backscattered electron (BSE) imaging shows promise for orthopaedic and bone research. BSE images of bone may be captured on-line directly from the scanning electron microscope (SEM), and then analyzed to produce a backscattered electron profile (BSEP), a modified image graylevel histogram which is representative of the mineral content in bone. The goals of this work were 1) develop a reproducible graylevel calibration technique for bone specimens, and 2) determine a conservative time interval during which SEM operating conditions would remain stable. Calibration standards containing pure aluminum and pure magnesium wires were placed in the SEM with human cancellous bone. Baseline imaging conditions were first established by adjusting the SEM until the bone image displayed good resolution and graylevel separation between regions of different mineral content. Microscope brightness and contrast controls were randomly changed to initiate the new operating conditions of another imaging session, and graylevel values from the calibration metals were used to readjust the microscope back to baseline operating conditions. Weighted mean graylevel values of the BSEPs from calibration trials were compared to those of the baseline. Data showed that bone images could be reproduced within 1.2 percent. It was also concluded that our equipment required calibration checks at 20 minute intervals.