Raman-derived compositional properties, including mineralization, mineral crystallinity, and carbonation, are important predictors of bone mechanical functions.12,14,28 Studies on animal and human bones revealed that these Raman parameters are indicative of bone quality alterations associated with age and disease at tissue level.29–31 Collagen mineralization indicates the amount of bone minerals, mainly consisting of calcium phosphate in an apatite crystalline structure, deposited in collagen matrix.32,33 Hydroxyapatite crystallinity is an indicator for the size and stoichiometric perfection of the deposited mineral crystal.34 Both properties have been found to increase with tissue age and demonstrated reduced heterogeneity within aged tissue, along with deteriorating mechanical properties.12,29,31 Since bone is a birefringent material, the polarization of incident light and molecular orientation in the tissue can also affect the intensities of Raman bands.35–38 The major vibration of phosphate is along the direction of collagen fibrils, while the carbonyl groups (amide I) of collagen mainly stretch perpendicular to the fibrils. Therefore, both bands are very sensitive to sample orientation and the instrument polarization. The phase mismatch between phosphate and amide I could introduce uncertainty when interpreting the difference of phosphate between groups. As a result, other Raman bands, such as phosphate and , amide III, and proline, could be essential quality control parameters for compositional analysis.35,37 The area ratio of phosphate to amide III has been reported as insensitive to sample orientation, indicating phosphate is more relevant to the mineralization than phosphate .36,37 Since the intensities of phosphate and proline change with phase matching under polarized light, their ratio (phosphate ) has also been recommended for the calculation of mineralization.35 In the current study, both phosphate and phosphate have been investigated and demonstrated consistent outcomes at the metaphysis region (positions at 0 and 500 μm). The significance of differences ( value) between the two groups in phosphate is not as dramatic as those in phosphate at the same position, indicating that there might be variations in the orientation of collagen fibrils besides mineralization in the tumor-bearing bones.