Skip to main content Accessibility help

Tunable glass reference materials for quantitative backscattered electron imaging of mineralized tissues

  • Sara E. Campbell (a1), Roy H. Geiss (a2), Steve A. Feller (a3) and Virginia L. Ferguson (a4)


Backscattered electron microscopy provides gray-level contrast resulting from variations in atomic composition. Through the use of reference materials, quantitative backscattered electron (qBSE) imaging can be used to measure the mineral content of mineralized tissues at submicron resolution. We have developed novel tunable reference materials that can be adjusted for analysis of an individual tissue or a wide range of tissues with variable atomic density. As an alternative to conventional metallic reference materials, these amorphous materials maintain long-term stability and possess no long-range order that may induce channeling contrast. Using these reference materials, we characterized the mineral content of a broad range of mineralized tissues from immature mouse femur to whale bulla. Mineral volume fraction correlated to more traditional measurements of mineral content with microcomputed tomography and ashing techniques. Further, we demonstrate the advantage of location-matched measurements of nanomechanical properties and qBSE mineral content.


Corresponding author

a)Address all correspondence to this author. e-mail:


Hide All
1.Ferguson, V.L., Bushby, A.J., and Boyde, A.: Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J. Anat. 203(2), 191 (2003).
2.Bloebaum, R.D., Skedros, J.G., Vajda, E.G., Bachus, K.N., and Constantz, B.R.: Determining mineral content variations in bone using backscattered electron imaging. Bone 20(5), 485 (1997).
3.Boyde, A., Elliott, J., and Jones, S.: Stereology and histogram analysis of backscattered electron images - age-changes in bone. Bone 14(3), 205 (1993).
4.Roschger, P., Fratzl, P., Eschberger, J., and Klaushofer, K.: Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies. Bone 23(4), 319 (1998).
5.Skedros, J.G., Bloebaum, R.D., Bachus, K.N., Boyce, T., and Constantz, B.: Influence of mineral-content and composition on graylevels in backscattered electron images of bone. J. Biomed. Mater. Res. 27(1), 57 (1993).
6.Angker, L., Nockolds, C., Swain, M.V., and Kilpatrick, N.: Correlating the mechanical properties to the mineral content of carious dentine - a comparative study using an ultra-micro indentation system (UMIS) and SEM-BSE signals. Arch. Oral Biol. 49(5), 369 (2004).
7.Sinclair, K.D., Curtis, B.D., Koller, K.E., and Bloebaum, R.D.: Characterization of the anchoring morphology and mineral content of the anterior cruciate and medial collateral ligaments of the knee. Anat. Rec. 294(5), 831 (2011).
8.Kingsmill, V.J., Boyde, A., Davis, G.R., Howell, P.G.T., and Rawlinson, S.C.F.: Changes in bone mineral and matrix in response to a soft diet. J. Dent. Res. 89(5), 510 (2010).
9.Tjhia, C.K., Odvina, C.V., Rao, D.S., Stover, S.M., Wang, X., and Fyhrie, D.P.: Mechanical property and tissue mineral density differences among severely suppressed bone turnover (SSBT) patients, osteoporotic patients, and normal subjects. Bone 49(6), 1279 (2011).
10.Zebaze, R.M.D., Jones, A.C., Pandy, M.G., Knackstedt, M.A., and Seeman, E.: Differences in the degree of bone tissue mineralization account for little of the differences in tissue elastic properties. Bone 48(6), 1246 (2011).
11.Smith, L.J., Schirer, J.P., and Fazzalari, N.L.: The role of mineral content in determining the micromechanical properties of discrete trabecular bone remodeling packets. J. Biomech. 43(16), 3144 (2010).
12.Fratzl-Zelman, N., Morello, R., Lee, B., Rauch, F., Glorieux, F.H., Misof, B.M., Klaushofer, K., and Roschger, P.: CRTAP deficiency leads to abnormally high bone matrix mineralization in a murine model and in children with osteogenesis imperfecta type VII. Bone 46(3), 820 (2010).
13.Boyce, T.M., Bloebaum, R.D., Bachus, K.N., and Skedros, J.G.: Reproducible method for calibrating the backscattered electron signal for quantitative assessment of mineral-content in bone. Scanning Microsc. 4(3), 591 (1990).
14.Traini, T., Degidi, M., Iezzi, G., Artese, L., and Piattelli, A.: Comparative evaluation of the peri-implant bone tissue mineral density around unloaded titanium dental implants. J. Dent. 35(1), 84 (2007).
15.Reid, S.A. and Boyde, A.: Changes in the mineral density distribution in human bone with age: Image analysis using backscattered electrons in the SEM. J. Bone Miner. Res. 2(1), 13 (1987).
16.Lloyd, G.: Atomic-number and crystallographic contrast images with the SEM - a review of backscattered electron techniques. Mineral. Mag. 51(359), 3 (1987).
17.Howell, P.G.T., Davy, K.M.W., and Boyde, A.: Mean atomic number and backscattered electron coefficient calculations for some materials with low mean atomic number. Scanning 20(1), 35 (1998).
18.Boyde, A. and Jones, S.J.: Back-scattered electron imaging of skeletal tissues. Metab. Bone. Dis. Relat. 5(3), 145 (1983).
19.Howell, P.G.T. and Boyde, A.: Monte Carlo simulations of electron scattering in bone Bone 15(3), 285 (1994).
20.Goldstein, J., Newbury, D., Joy, D., Layman, C., Echlin, P., Lifshin, E., Sawyer, L., and Michael, J.: Scanning Electron Microscopy and X-ray Microanalysis, 3rd ed. (Springer, New York, 2003).
21.Oyen, M.L., Ferguson, V.L., Bembey, A.K., Bushby, A.J., and Boyde, A.: Composite bounds on the elastic modulus of bone. J. Biomech. 41(11), 2585 (2008).
22.Vajda, E.G., Skedros, J.G., and Bloebaum, R.D.: Consistency in calibrated backscattered electron images of calcified tissues and minerals analyzed in multiple imaging sessions. Scanning Microsc. 9(3), 741 (1995).
23.Islam, M.M., Holland, D., and Scales, C.R.: Chemical durability and conductivity of mixed borosilicate glasses for high level waste immobilisation. Phys. Chem. Glasses 49(5), 229 (2008).
24.Ewing, R.C.: Nuclear waste form glasses: The evaluation of very long-term behaviour. Mater. Technol. 16(1), 30 (2001).
25.Mullenbach, T., Franke, M., Ramm, A., Betzen, A.R., Kapoor, S., Lower, N., Munhollon, T., Berman, M., Affatigato, M., and Feller, S.A.: Structural characterisation of alkaline earth borosilicate glasses through density modelling. Phys. Chem. Glasses 50(2), 89 (2009).
26.Feller, S., Lodden, G., Riley, A., Edwards, T., Croskrey, J., Schue, A., Liss, D., Stentz, D., Blair, S., Kelley, M., Smith, G., Singleton, S., Affatigato, M., Holland, D., Smith, M.E., Kamitoss, E.I., Varsamis, C.P.E., and Ioannou, E.: A multispectroscopic structural study of lead silicate glasses over an extended range of compositions. J. Non-Cryst. Solids 356(6–8), 304 (2010).
27.Joy, D.C.: A database on electron-solid interactions. Scanning 17(5), 270 (1995).
28.Arnal, F., Verdier, P., and Vincensini, P.: Backscattering coefficient in the case of monoenergetic electrons arriving at the target at an oblique incidence. Compt. Rend.,Ser. B 268, 1526 (1969).
29.Heinrich, K.: Theory of quantitative electron probe microanalysis, in Electron Beam X-ray microanalysis, Heinrich, K., ed. (Van Nostrand Reinhold, New York, 1981); pp. 219254.
30.Herrmann, R. and Reimer, L.: Backscattering coefficient of multicomponent specimens. Scanning 6(2), 20 (1984).
31.Reuter, W.: The ionization function and its application to the electron probe analysis of thin films, in Proceedings of Sixth International Conference On X-Ray Optics and Microanalysis, edited by Shinoda, G., Kohra, K., and Ichinokawa, T. (University of Tokyo Press, Tokyo, Japan, 1972); pp. 121130.
32.Castaing, R.: Electron probe microanalysis. Adv. Electron. Electron Phys. 13, 317 (1960).
33.Weast, R.: Handbook of Chemistry and Physics, 57th ed. (CRC Press Inc., Cleveland, OH, 1976).
34.Wong, F.S.L. and Elliott, J.C.: Theoretical explanation of the relationship between backscattered electron and x-ray linear attenuation coefficients in calcified tissues. Scanning 19(8), 541 (1997).
35.Elliott, J.C.: Calcium phosphate biominerals, in Phosphates: Geochemical, Geobiological, and Materials Importance, edited by Kohn, M.J., Rakovan, J., and Hughes, J.M. (Mineralogical Society America, Wahington DC, 2002); pp. 427.
36.Parfitt, A.M., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M., and Recker, R.R.: Bone histomorphometry - standardization of nomenclature, symbols, and units. J. Bone Miner. Res. 2(6), 595 (1987).
37.Oliver, W.C. and Pharr, G.M.: An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J. Mater. Res. 7(6), 1564 (1992).
38.Bushby, A.J.: Nano-indentation using spherical indenters. Nondestr. Test. Eval. 17(4), 213 (2001).
39.Tesch, W., Eidelman, N., Roschger, P., Goldenberg, F., Klaushofer, K., and Fratzl, P.: Graded microstructure and mechanical properties of human crown dentin. Calcified Tissue Int. 69(3), 147 (2001).
40.Rho, J.Y., Roy, M.E., Tsui, T.Y., and Pharr, G.M.: Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. J. Biomed. Mater. Res. 45(1), 48 (1999).
41.Gupta, H.S., Stachewicz, U., Wagermaier, W., Roschger, P., Wagner, H.D., and Fratzl, P.: Mechanical modulation at the lamellar level in osteonal bone. J. Mater. Res. 21(8), 1913 (2006).
42.Currey, J.D.: Three analogies to explain the mechanical properties of bone. Biorheology 2, 1 (1964).
43.Katz, J.L.: Hard tissue as a composite material .1. bounds on elastic behavior. J. Biomech. 4(5), 455 (1971).
44.Hashin, Z. and Shtrickman, S.: A variational approach to the theory of the elastic behaviour of multiphase materials. J. Mech. Phys. Solids 11, 127 (1963).
45.Herakovich, C.T.: Mechanics of Fibrous Composites (Wiley, New York, NY, 1997).


Tunable glass reference materials for quantitative backscattered electron imaging of mineralized tissues

  • Sara E. Campbell (a1), Roy H. Geiss (a2), Steve A. Feller (a3) and Virginia L. Ferguson (a4)


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed