Influence of topography and specimen preparation on backscattered electronimages of bone

Backscattered electron (BSE) images of bone exhibit graylevel contrast betw een adjacent lamellae. Mathematical models suggest that interlamellar contr ast in BSE images is an artifact due to topographic irregularities. However , little experimental evidence has been published to support these models, and it is not clear whether submicron topographical features will alter BSE graylevels. The goal of this study was to determine the effects of topogra phy on BSE image mean graylevels and graylevel histogram widths using conve ntional specimen preparation techniques. White-light interferometry and qua ntitative BSE imaging were used to investigate the relationship between the BSE signal and specimen roughness. Backscattered electron image graylevel histogram widths correlated highly with surface roughness in rough preparat ions of homogeneous materials. The relationship between BSE histogram width and surface roughness was specimen dependent. Specimen topography coincide d with the lamellar patterns within the bone tissue. Diamond micromilling r educed average surface roughness when compared with manual polishing techni ques but did not significantly affect BSE graylevel histogram width. The st udy suggests that topography is a confounding factor in quantitative BSE an alysis of bone. However, there is little quantitative difference between lo w-to-moderate magnification BSE images of bone specimens prepared by conven tional polishing or diamond micromilling.