Separation of the Raman spectral signatures of bioapatite and collagen in compact mouse bone bleached with hydrogen peroxide

We studied the effectiveness of hydrogen peroxide in removing visible laser-induced photoluminescence from the Raman spectra of compact bone of mice. In testing various bone sample preparations, we found that hydrogen peroxide bleaching was most effective when applied directly to fresh or fresh-frozen bone samples. The extent of the reduction in photoluminescence in the peroxide-bleached bone was such that the Raman spectrum could be readily recorded with 532-nm laser excitation. A comparison of bone samples before and after hydrogen peroxide bleaching shows that the Raman shifts of all the collagen and bioapatite bands are unaffected by the peroxide bleach. Moreover, the low spectral backgrounds of the peroxide-treated bone samples permit the Raman spectra of these two major components of bone to be fully separated. The Raman spectrum of collagen-rich periosteum was subtracted from the Raman spectrum of compact bone, which isolated the Raman spectral signature of the bioapatite fraction of the bone. This derived spectrum of bioapatite was then used, through spectral subtraction, to generate the spectral signature of the collagen component of the bone. All the major and minor Raman bands of collagen and bioapatite can be identified in these separate spectra.