Imaging the material properties of bone specimens using reflection-based infrared microspectroscopy - PubMed (original) (raw)

. 2012 Apr 17;84(8):3607-13.

doi: 10.1021/ac203375d. Epub 2012 Apr 4.

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

Alvin S Acerbo et al. Anal Chem. 2012.

Abstract

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.

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Figures

Figure 1

Figure 1

(Red) A typical FTIRM spectrum of bone showing the characteristic protein and mineral components. (Blue) A FTIRM spectrum taken from a bone section that is too thick, illustrating detector saturation at the ν1ν3 phosphate peak. (Black) The FTIR spectrum of the embedding medium, PMMA, showing peaks that overlap with the bone spectrum. Spectra were offset by 0.5 absorbance units for clarity.

Figure 2

Figure 2

(A) A FTIRM spectrum of a mineralized bone block collected in a reflection geometry. (B) FTIRM absorbance spectra calculated from the reflectance data in (A) with a low-frequency cutoff at 650 (black), 400 (red) and 70 cm−1 (blue). Spectra were offset by 0.25 absorbance units for clarity.

Figure 3

Figure 3

(A) FTIRM reflectance spectra of a polished (red) and unpolished (black) bone block. The polished bone block has a net reflectance nearly double that of the unpolished block over the entire range of the spectrum. (B) FTIRM absorbance spectra calculated from the reflectance data in (A). The polished bone block (red) has a higher absorbance than the unpolished block (black), consistent with the increased reflectance as seen in (A). However, the relative peak intensities are not affected by the quality of polishing.

Figure 4

Figure 4

(A) Schematic overview of the reflection geometry configuration using a Schwarzschild objective typically employed in IR microscopes. The central obscuration limits the half angle to a range between 15 - 40°. (B) Plot of the reflection coefficient versus angle of incidence as a function of polarization for hydroxyapatite (n=1.530). The reflection coefficient changes as a function of angle of incidence, but remains sufficiently constant out to 40° such that is does not measurably affect analysis.

Figure 5

Figure 5

(A) Schematic of specimen preparation. Thin sections were cut from the top surface of the bone block and imaged in a transmission geometry. The bone block was then imaged before and after polishing. (B) Light micrograph of an embedded and polished bone specimen with the highlighted area showing the region that was scanned using reflection FTIRM. (C) Integration maps showing the distribution of mineralization, crystallinity, carbonate substitution and collagen cross-linking for matching bone blocks and thin sections.

Figure 6

Figure 6

(A) Transmission (red) and reflection-derived (blue) absorbance spectra and second-derivative spectra showing identical peak positions of the amide I, amide II, and CO 2-3 bands. Curve-fitting of the ν1,ν3 PO43− domain from (B) transmission and (C) reflection spectra based on peak positions from second-derivative spectra. Individual Gaussian/Lorentzian distributions and the resultant spectrum are also shown (black). The stoichiometric and non-stoichiometric apatite peaks are shifted from 1032 and 1021 cm−1 in the transmission spectrum to 1035 and 1025 cm−1 in the reflection-derived absorbance spectrum.

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