The spectrum of bone consists of peaks from two components: (1) the inorganic mineral component, which is a calcium phosphate that is similar—but by no means identical—to hydroxylapatite,11 (link),12 and (2) a macromolecular organic component that is dominated by type I collagen. Since the nanometer-sized mineral crystals are intimately associated with the organic collagen fibers, the spectra of these two components cannot be acquired separately. The size of the apatite crystals (platelets of about ~50 nm × ~50 nm that are only several nanometers thick13 ) is below the spatial resolution of visible light. Thus, the spatial resolution of the Raman microprobe technique (~1 μm) can only provide a “bulk” analysis of bone, and a spectrum obtained on bone will always include the peaks that are due to both the mineral component and the organic component.
Visible Raman spectroscopy on biological tissues, especially of cellular components, is often problematic due to intrinsic fluorescence.14 (link) However, dense collagenous tissues such as tendon and bone can be examined successfully even with 532 nm excitation.15 (link) In our minimally prepared samples, the Raman peaks for both the mineral and the organic components of bone were superimposed on a broad, featureless, steadily increasing fluorescence background (Fig. 2, top). Baseline correction for each individual spectrum was performed via an operator-chosen 10-to-15-point leveling operation with linear curve fits between neighboring points (see result in Fig. 2, bottom). This careful background correction allowed not only for a better comparison of individual spectra in figures but also permitted more precise quantitative interpretations of the spectra. Peaks were deconvolved with a mixed Gaussian–Lorentzian algorithm after individual spectra were baseline corrected. All spectral processing (i.e., baseline correction, deconvolutions, peak area calculations) was performed with the Grams/32R software (Galactic, Salem, NH).