Quantitative Backscattered Electron Imaging of Bone Using a Thermionic or a Field Emission Electron Source

• Published in: Calcified tissue international 2021-08, Vol.109 (2), p.190-202
• Main Authors: Hartmann, Markus A., Blouin, Stéphane, Misof, Barbara M., Fratzl-Zelman, Nadja, Roschger, Paul, Berzlanovich, Andrea, Gruber, Gerlinde M., Brugger, Peter C., Zwerina, Jochen, Fratzl, Peter
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Bone density; Bone imaging; Bone mineral content; Calcium; Cell Biology; Electric fields; Endocrinology; Hydroxyapatite; Life Sciences; Mineralization; Original Research; Orthopedics; Osteoporosis; Scanning electron microscopy; Spatial discrimination; Tungsten; X-rays
• ISSN: 0171-967X; 1432-0827
• DOI: 10.1007/s00223-021-00832-5

Quantitative backscattered electron imaging is an established method to map mineral content distributions in bone and to determine the bone mineralization density distribution (BMDD). The method we applied was initially validated for a scanning electron microscope (SEM) equipped with a tungsten hairpin cathode (thermionic electron emission) under strongly defined settings of SEM parameters. For several reasons, it would be interesting to migrate the technique to a SEM with a field emission electron source (FE-SEM), which, however, would require to work with different SEM parameter settings as have been validated for DSM 962. The FE-SEM has a much better spatial resolution based on an electron source size in the order of several 100 nanometers, corresponding to an about 10 5 to 10 6 times smaller source area compared to thermionic sources. In the present work, we compare BMDD between these two types of instruments in order to further validate the methodology. We show that a transition to higher pixel resolution (1.76, 0.88, and 0.57 μm) results in shifts of the BMDD peak and BMDD width to higher values. Further the inter-device reproducibility of the mean calcium content shows a difference of up to 1 wt% Ca, while the technical variance of each device can be reduced to ± 0.17 wt% Ca. Bearing in mind that shifts in calcium levels due to diseases, e.g., high turnover osteoporosis, are often in the range of 1 wt% Ca, both the bone samples of the patients as well as the control samples have to be measured on the same SEM device. Therefore, we also constructed new reference BMDD curves for adults to be used for FE-SEM data comparison.