The meaning of graylevels in backscattered electron images of bone

Backscattered electron (BSE) imaging is considered to be a useful technique for determining relative differences in bone tissue density. However, it is not clear how graylevel variations seen in BSE images of bone tissue, which are primarily dependent on the tissue's average atomic number, correlate to tissue density (g/cm3) and mineral content. Simulated bone tissues, ranging from 32-50% mineral by volume, were made by mixing synthetic hydroxyapatite with a simulated organic matrix. This technique allowed mineral content to be varied while mineral composition and crystallography remained constant. The densities of the simulated tissues were determined using Archimedes' principle. Average atomic numbers of the simulated tissues were interpolated from a regression of BSE graylevel against average atomic numbers of pure standard materials. A strong positive correlation was found to exist between mineral content and density (r2 = 0.978) as well as between mineral content and atomic number (r2 = 0.965). The average graylevel in the BSE image also exhibited a positive correlation to mineral content (r2 = 0.965) and density (r2 = 0.923). Graylevel variations in BSE images of simulated bone tissue were shown to be strongly correlated to density and mineral content, but only as a coincidence of their association with atomic number.