Howell P G, Boyde A
Department of Anatomy and Developmental Biology, University College, London, UK.
Bone. 1994 May-Jun;15(3):285-91. doi: 10.1016/8756-3282(94)90290-9.
Relative changes in the mineralization level within bone can be studied using backscattered electron (BSE) imaging in a scanning electron microscope (SEM). We calculated the size and shape of the volume element studied, choosing conditions which are typical for practical experimental work with polymethylmethacrylate (PMMA)-embedded bone. Absolutely flat surfaces of embedded bone blocks cannot be generated, and a further aim was to examine the effect of the surface topography on the detected BSE signal level. For normal beam incidence, 20 kV, and modeling an annular detector by collecting BSE with take-off angles of between 45 degrees and 75 degrees to the flat sample surface, it was found that the collectable BSE signal intensity peaks for electrons which leave the specimen surface at a radial distance of approximately 1 micron from the beam impact point. The layered structure of the bone generates topographic relief on polishing. Modeling this by a sinusoidal profile of wavelength 5.0 and amplitude 0.5 micron and again for 20 kV, it was found that the signal derived from the troughs is reduced by 14.4% and that from the crests is increased by 17.2%. The two effects may add constructively to generate the frequently observed strong contrast correlating with the distribution of bone lamellae. No net change in mineral packing density would be expected from a change in collagen orientation, and the lamellar contrast observed in practice can be explained solely by the topographic contrast.
