A NEW SCANNING ELECTRON-MICROSCOPY APPROACH TO THE QUANTIFICATION OF BONE-MINERAL DISTRIBUTION - BACKSCATTERED ELECTRON IMAGE GREY-LEVELS CORRELATED TO CALCIUM K-ALPHA-LINE INTENSITIES

The introduction of backscattered electron (BSE) imaging in scanning e lectron microscopy (SEM) has led to new possibilities for the evaluati on of mineral distributions in bone on a microscopic level. The differ ent grey-levels seen in the BSE-images can be used as a measure for th e local mineral content of bone. In order to calibrate these BSE-grey- levels (BSE-GL) and correlate them to mineral contents, various attemp ts, using reference samples with known weighted mean atomic number and /or using simulated bone tissues with known hydroxyapatite concentrati ons, have been made. In contrast, a new approach is presented here bas ed on measurements of the X-ray intensities of the calcium K alpha-lin e on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlati on between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. Th e resulting mineral concentration histograms have a dynamic range from 0 to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The pre sented modifications of the BSE method strongly enhance its feasibilit y in the field of bone research and its application as a special diagn ostic tool for bone diseases.