Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the phosphate-to-amide I ratio (a measure of the mineral to collagen ratio) can vary by as much as 8%, and the phosphate-to-carbonate ratio (a measure of carbonate inclusion in mineral crystals) by as much as 5%. The data are preliminary but are important because they reveal a spatial variation at a scale that is much larger than many of the spectral maps reported in the literature to date. Thus they illustrate natural variation in chemical composition that could have been overlooked in such studies or could have appeared as an undue error where the overall composition of the bone was investigated. Quantifying the variation in mid-shaft cortical bone at the millimeter/centimeter scale reduces the possibility of natural heterogeneity obscuring the average bone composition, or being mistaken for experimental signal, and results in an improvement in the sampling accuracy analogous to that obtained by switching from micrometer-size point spectra of bones to spectral images obtained across hundreds of micrometers. Although the study was carried out using Raman spectroscopy, the underlying cause of the variation is ascribed to the variation of the chemical composition of the bone; therefore the findings have direct implications for other chemically specific analytical methods such as Fourier transform infrared spectroscopy or nuclear magnetic resonance spectroscopy.
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