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 (mu)m 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 stoichiometriconstoichiometric 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.
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机译：傅里叶变换红外光谱（FTIRM）是一种用于绘制骨骼和其他矿化组织的材料特性（包括矿化，结晶度，碳酸盐替代和胶原交联）的材料的方法。传统上，此技术是在基于传输的几何体中执行的，这需要准备塑料嵌入的薄型截面，从而限制了其功能。在这里，我们在理论上和经验上证明了基于反射的FTIRM的发展，它是广泛采用的基于透射的FTIRM的替代产品，它减少了标本的制备时间并拓宽了可成像标本的范围。在这项研究中，将成熟的小鼠股骨包埋在塑料中，并将纵切面切成4微米的厚度，以进行基于透射的FTIRM测量。其余的骨块在与透射部分紧邻的区域上进行抛光，以进行基于镜面反射的FTIRM测量。反射率数据的Kramers-Kronig分析得出介电响应，可直接确定吸收系数。使用薄部分的透射光谱，根据经验验证反射率衍生的吸光度。矿化，碳酸盐替代和胶原交联的光谱分配在透射和反射几何中是无法区分的，而化学计量/非化学计量的磷灰石结晶度参数从透射型的1032/1021 cm〜（-1）变为1035/1025 cm 〜（-1）在基于反射的数据中。这种基于反射的FTIRM的理论论证和经验验证消除了对骨骼薄片的需求，并且更容易促进了与其他方法（例如同一样品的纳米压痕和定量背散射电子成像（qBSE））的直接关联。它提供了一个独特的框架来关联骨骼的材料和机械特性。
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