Raman microspectroscopy and quantitative backscattered electron imaging (qBEI) of bone are powerful tools to investigate bone material properties. Both methods provide information on the degree of bone matrix mineralization. However, a head-to-head comparison of these outcomes from identical bone areas has not been performed to date. In femoral midshaft cross sections of three women, 99 regions (20×20 μm2) were selected inside osteons and interstitial bone covering a wide range of matrix mineralization. As the focus of this study was only on regions undergoing secondary mineralization, zones exhibiting a distinct gradient in mineral content close to the mineralization front were excluded. The same regions were measured by both methods. We found a linear correlation (R2=0.75) between mineral/matrix as measured by Raman spectroscopy and the wt. %Mineral/(100-wt. %Mineral) as obtained by qBEI, in good agreement with theoretical estimations. The observed deviations of single values from the linear regression line were determined to reflect biological heterogeneities. The data of this study demonstrate the good correspondence between Raman and qBEI outcomes in describing tissue mineralization. The obtained correlation is likely sensitive to changes in bone tissue composition, providing an approach to detect potential deviations from normal bone.
Fourier transform infrared imaging (FTIR) and Raman Microspectroscopy are powerful tools for characterizing the
distribution of different chemical moieties in heterogeneous materials. FTIR and Raman measurements have been
adapted to assess the maturity of the mineral and the quality of the organic component (collagen and non-collagenous
proteins) of the mineralized tissue in bone. Unique to the FTIRI analysis is the capability to provide the spatial
distribution of two of the major collagen cross-links (pyridinoline, and dehydro-dihydroxylysinonorleucine) and through
the study of normal and diseased bone, relate them to bone strength. These FTIR parameters have been validated based
on analysis of model compounds. It is widely accepted that bone strength is determined by bone mass and bone quality.
The latter is a multifactorial term encompassing the material and structural properties of bone, and one important aspect
of the bone material properties is the organic matrix. The bone material properties can be defined by parameters of
mineral and collagen, as determined by FTIR and Raman analysis. Considerably less attention has been directed at
collagen, although there are several publications in the literature reporting altered collagen properties associated with
fragile bone, in both animals and humans. Since bone is a heterogeneous tissue due to the remodeling process,
microscopic areas may be carefully selected based on quantitative Backscattered Electron Imaging or histological
staining, thus ensuring comparison of areas with similar metabolic activity and mineral content. In conclusion, FTIRI
and Raman vibrational spectroscopy are proving to be powerful tools in bone-related medical research.