Errors in quantitative backscattered electron analysis of bone standardized by energy‐dispersive x‐ray spectrometry

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Abstract
Backscattered electron (BSE) imaging has proven to be a useful method for analyzing 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 suggested that BSE images can be standardized by energy‐dispersive x‐ray spectrometry (EDX). Unfortunately, EDX‐standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX‐standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methyl‐methacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.