Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive X-ray spectrometry

Backscattered electron (BSE) imaging has proven to be a useful method for a nalyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has s uggested that BSE images can be standardized by energy-dispersive x-ray spe ctrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underes timate the mineral content of bone when compared with traditional ash measu rements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of anal ytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. Th e primary source of error was found to be inaccurate ZAF corrections to acc ount for the organic phase of the bone matrix. Conductive coatings, methylm ethacrylate embedding media, and minor elemental constituents in bone miner al introduced negligible errors. It is suggested that the errors would rema in constant and an empirical correction could be used to account for the de ficit. However, extensive preliminary testing of the analysis equipment is essential.