Polarization control of Raman spectroscopy optimizes the assessment of bone tissue

There is potential for Raman spectroscopy (RS) to complement tools for bone diagnosis due to its ability to assess compositional and organizational characteristics of both collagen and mineral. To aid this potential, the present study assessed specificity of RS peaks to the composition of bone, a birefringent material, for different degrees of instrument polarization. Specifically, relative changes in peaks were quantified as the incident light rotated relative to the orientation of osteonal and interstitial tissue, acquired from cadaveric femurs. In a highly polarized instrument (10(6)∶1 extinction ratio), the most prominent mineral peak (ν1 Phosphate at 961 cm(-1)) displayed phase similarity with the Proline peak at 856 cm(-1). This sensitivity to relative orientation between bone and light observed in the highly polarized regime persisted for certain sensitive peaks (e.g., Amide I at 1666  cm(-1)) in unaltered instrumentation (200∶1 extinction ratio). Though Proline intensity changed with bone rotation, the phase of Proline matched that of ν1 Phosphate. Moreover, when mapping ν1 Phosphate/Proline across osteonal-interstitial borders, the mineralization difference between the tissue types was evident whether using a 20x or 50x objectives. Thus, the polarization bias inherent in commercial RS systems does not preclude the assessment of bone composition when using phase-matched peaks.