Quantitative backscattered electron imaging (qBEI) at the scanning electron microscope (SEM) was developed for the investigation of the mineralization pattern of bone samples and other mineralized tissues. The information on the local variation of the calcium concentration within the bone sample is essential since the amount of mineral and its distribution within the specimen are important contributors to the mechanical quality of the bone.
For the investigation by qBEI, the unmineralized sample has to be embedded into polymethylmethacrylate (PMMA). The block samples are then polished and carbon coated. In the SEM, the surface of the sample is scanned by the electron beam and the intensity of the backscattered electron (BE)-signal from each measured site is recorded.
This BE-signal is mainly determined by the local calcium concentration of the specimen, as reflected by different gray-scales in the image. High calcium concentration can be identified by its bright gray-level in the image, low calcium concentration by its dark gray-level. The embedding material PMMA appears black in this image.
From the BE-gray-level images, histograms can be obtained which are displaying the percentage of the sample occupied by a specific gray-level. If the gray-levels are transferred to Ca-values, the bone samples can be analyzed quantitatively for their calcium concentrations by evaluating the bone mineral density distribution (BMDD). The accuracy of this technique (the minimum difference in calcium concentration that can be resolved by the method) is 0.17wt% Ca (weight-percent calcium). It was shown that the BMDD of trabecular bone from healthy adult individuals is similar independent of age, gender, ethnic origin and skeletal site (vertebra, patella, femoral neck, iliac crest), which is a strong indication for the optimization of the mineral pattern of bone. In the case of diseases, deviations from the “normal” BMDD could be observed and therefore the BMDD has become a powerful tool for the diagnosis of specific bone diseases and can be used as an indicator for the effectiveness of treatments.
For the comparison of a measured BMDD with the “normal” BMDD from healthy individuals, several parameters are obtained from the distribution and compared to the reference-values, such as the peak position CaPeak (which displays the most frequently occurring calcium concentration within the studied bone area) and CaMean (which is the weighted mean calcium concentration).
Additionally, CaWidth, the width of the distribution (full width at half maximum) which is reflecting the homogeneity of mineralization (the lower this parameter, the more homogeneous is the mineralization pattern of the sample) and CaLow (a parameter which displays the percentage of calcium concentrations below 17.68 wt% Ca referring to the amount of newly formed bone matrix) can be deduced from the BMDD and compared to the normal values of these parameters.