Compositional assessment of bone by Raman spectroscopy

Abstract

Raman spectroscopy (RS) is used to analyze the physiochemical properties of bone because it is non-destructive and requires minimal sample preparation. With over two decades of research involving measurements of mineral-to-matrix ratio, type-B carbonate substitution, crystallinity, and other compositional characteristics of the bone matrix by RS, there are multiple methods to acquire Raman signals from bone, to process those signals, and to determine peak ratios including sub-peak ratios as well as the full-width at half maximum of the most prominent Raman peak, which is nu1 phosphate (ν₁PO₄). Selecting which methods to use is not always clear. Herein, we describe the components of RS instruments and how they influence the quality of Raman spectra acquired from bone because signal-to-noise of the acquisition and the accompanying background fluorescence dictate the pre-processing of the Raman spectra. We also describe common methods and challenges in preparing acquired spectra for the determination of matrix properties of bone. This article also serves to provide guidance for the analysis of bone by RS with examples of how methods for pre-processing the Raman signals and for determining properties of bone composition affect RS sensitivity to potential differences between experimental groups. Attention is also given to deconvolution methods that are used to ascertain sub-peak ratios of the amide I band as a way to assess characteristics of collagen type I. We provide suggestions and recommendations on the application of RS to bone with the goal of improving reproducibility across studies and solidify RS as a valuable technique in the field of bone research.

Figure 1:. A schematic depiction of a Raman micro-spectroscopy instrument.

A) A Raman spectroscopy instrument has 4 primary components: a light source, a stage for the sample being analyzed, optics, and a detector. B) A commercial RS instrument includes mirrors, optical filters, focusing lenses, and objective lens to deliver the laser onto the sample and guide the collection of Raman scattered photons to the spectrometer (i.e., grating and detector, which is a charged-coupled device or CCD). The grating separates photons according to their wavelength in space so that the pixels of the CCD captures their intensity. Raman micro-spectroscopy has a confocality option which is provided by a pin hole aperture and slit. The optics of Raman micro-spectroscopy can preserve the polarization axis of the laser.

Figure 2.. Effect of focusing on peak intensity.

Raman spectra were acquired from a human cortical bone using a 785 nm laser source under a well-focused laser (black), improperly focused laser (red) and defocused laser (blue). When the laser is not properly focused onto the unpolished bone surface, the intensity of peaks such as ν₁PO₄ (inset) is considerably lower.

Figure 3.. Effect of improper calibration of the Raman shift axis on peak locations.

Raman spectra were acquired from silicon using an 830 nm laser source before (red) and after (black) calibration (B). When a commercial research-grade RS instrument is not properly calibrated, the wavenumber location of peaks such as ν₁PO₄ (inset) are shifted from their known location (B).

Figure 4.. Effect rotating bone 90° relative to the polarization axis of the laser on Raman peaks of human cortical bone.

The diode laser is preferentially polarized such that the orientation of the light waves is narrowly distributed about an axis unlike unpolarized light or fully polarized light (A). By knowing the direction of the polarization axis of the laser, a bone sample can be rotated 90° such that the orientation of the osteons is parallel (black) or perpendicular (red) to the polarization direction (B). The Raman peaks of cadaveric cortical bone are higher when the osteons are parallel than when they are perpendicular to the polarization direction, but the change in height upon rotation is not the same across all peaks (C).

Figure 5.. Effect of acquisition time and number of accumulations on Raman spectrum of bone.

Raman spectra were acquired from the native unpolished human cortical bone obtained from a cadaveric femur using an 830 nm laser source while keeping the total acquisition time constant (e.g., scan time × number of accumulations = 60 s) but different combinations of scan time × number of accumulations. The spectral resolution of the Raman instrument was 1 cm⁻¹.

Figure 6.. Effect of polynomial order on the fit of the non-linear curve to the apparent baseline of a Raman spectrum of bone.

Because bone tissue contains fluorophores that auto-fluoresce upon exposure to laser light, all raw Raman spectra of bone have background fluorescence that obscures the relative of heights of various peaks. To remove or subtract the background fluorescence, a polynomial curve of some specified order (e.g., quartic) is fit to the apparent baseline of the Raman spectrum. Selecting too low of an order (e.g., a*x² + b*x + c) or too high of an order (a*x¹⁰ + b*x⁹ + c*x⁸ + d*x⁷ + e*x⁶ + f*x⁴ + g*x³ + h*x² + i*x² + j*x + k) under-fits certain regions of the spectrum or over-fits the polynomial baseline curve. The poor fit at the extreme ends of the Raman shift can be ignored if the region does not contain important peaks (i.e., can be truncated after removing background fluorescence).

Figure 7.. Differences in Raman spectra of bone due to noise filtering and different methods to determine peak ratios.

A fitted 5^th order polynomial curve subtracted the background fluorescence from the raw spectrum of human cortical bone, and then a S-G filter (4th order & window of 21) was (green) or was not (blue and offset) applied (A). Two typical methods for determining peak ratios include dividing one peak intensity (PI) height by another PI height or dividing an integrated area (IA) of a band by an IA of another band (B). *When using the PI method or IA method, the mineral peak is divided by proline peak or the combination of Pro and the hydroxyproline peak, respectively.

Figure 8.. Effect of baseline on crystallinity.

When a local linear baseline (red line) ‘corrects’ the height of the ν1 phosphate peak at ~960 cm⁻¹, the FWHM decreases meaning crystallinity increases (A). This linear correction (LC) also increases the correlation of determination (R²) of the fit of the Gaussian curve to the peak (B) when compared to a fit without correction (WC).

Figure 9.. Deconvolution procedure to fit sub-peaks to the amide I band.

Initial peak positions under the amide I envelope are based on the minima of the 2^nd derivative spectrum below zero (A). To deconvolve the sub-peaks, Gaussian functions are fit to the amide I band such that their location is close to initial location identified by the 2^nd derivative spectrum and their sum achieves a high coefficient of determination (R² > 0.990) (B). The spectrum in this example was baseline corrected using 5^th order polynomial fit, and a linear baseline was applied to the amide I band before the curve fitting. Origin Pro 8.5 was used for sub-peak finding and band fitting amide I.

Figure 10.. Sub-peak ratio correlations between 2 deconvolution methods.

When the sub-bands are allowed to move during the fitting process, 100% Gaussian curves do not produce sub-peak ratios that correlate with their corresponding sub-peak ratio as determined by a blend of Gaussian and Lorentzian functions (A). On the other hand, when fixing the position of the sub-bands to the local minima of the 2^nd derivative spectrum, the 2 different deconvolution methods determine sub-peak ratios that correlate, at least for 2 of the 3 ratios that were selected (B). This is not a justification for fixing the positions during the curve fits, but rather the choice of selecting Gaussian vs. Guassian/Lorentzian functions is less problematic when the positions of the sub-bands are fixed. P-value is provided for each Spearman’s correlation coefficient (r).

Figure 11.. A schematic depiction of a probe-based RS (Probe RS) instrument.

Probe RS instrument include fiber optics in a cable that passes the light from the excitation laser to the material of interest and the scattered light to the detector.