Imaging the material properties of bone specimens using reflection-based infrared microspectroscopy.

Fourier transform infrared microspectroscopy (FTIRM) is a widely used method for mapping the material properties of bone and other mineralized tissues, including mineralization, crystallinity, carbonate substitution, and collagen cross-linking. This technique is traditionally performed in a transmission-based geometry, which requires the preparation of plastic-embedded thin sections, limiting its functionality. Here, we theoretically and empirically demonstrate the development of reflection-based FTIRM as an alternative to the widely adopted transmission-based FTIRM, which reduces specimen preparation time and broadens the range of specimens that can be imaged. In this study, mature mouse femurs were plastic-embedded and longitudinal sections were cut at a thickness of 4 μm for transmission-based FTIRM measurements. The remaining bone blocks were polished for specular reflectance-based FTIRM measurements on regions immediately adjacent to the transmission sections. Kramers-Kronig analysis of the reflectance data yielded the dielectric response from which the absorption coefficients were directly determined. The reflectance-derived absorbance was validated empirically using the transmission spectra from the thin sections. The spectral assignments for mineralization, carbonate substitution, and collagen cross-linking were indistinguishable in transmission and reflection geometries, while the stoichiometric/nonstoichiometric apatite crystallinity parameter shifted from 1032/1021 cm(-1) in transmission-based to 1035/1025 cm(-1) in reflection-based data. This theoretical demonstration and empirical validation of reflection-based FTIRM eliminates the need for thin sections of bone and more readily facilitates direct correlations with other methods such as nanoindentation and quantitative backscatter electron imaging (qBSE) from the same specimen. It provides a unique framework for correlating bone's material and mechanical properties.