Raman spectroscopic imaging markers for fatigue-related microdamage in bovine bone

Raman spectroscopic markers have been determined for fatigue-related microd amage in bovine bone. Microdamage was induced using a cyclic fatigue loadin g regime. After loading, the specimens were stained en-bloc with basic fuch sin to facilitate damage visualization and differentiate fatigue-induced da mage from cracks generated during subsequent histological sectioning, Bone tissue specimens were examined by light microscopy and hyperspectral near-i nfrared Raman imaging microscopy, Three regions were defined-tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. R aman transects, lines of 150-200 Raman spectra, were used for initial tissu e surveys. Exploratory factor analysis of the transect Raman spectra has id entified spectroscopically distinct chemical microstructures of the bone sp ecimens that correlate with damage. In selected regions of damage, full hyp erspectral Raman images were obtained with 1.4-mu m spatial resolution. In regions of undamaged tissue, the phosphate vl band is found at 957 cm(-1), as expected for the carbonated hydroxyapatic bone mineral. However, in regi ons of visible microdamage, an additional phosphate nu(1) band is observed at 963 cm(-1) and interpreted as a more stoichiometric, less carbonated min eral species. Raman imaging confirms the qualitative relationship between t he Raman spectral signature of bone mineral and the type of microdamage in bovine bone. Two tentative explanations for the presence of less carbonated phosphate in damaged regions are proposed.