Soft-tissue spectral subtraction improves transcutaneous Raman estimates of murine bone strength in vivo

Transcutaneous determination of a bone's Raman spectrum is challenging because the type I collagen in the overlying soft tissue is spectroscopically identical to that in bone. In a previous transcutaneous study of murine tibiae, we developed a library-based model called SOLD to unmix spatially offset Raman measurements into three spectra: a bone estimate, a soft tissue estimate, and a residual. Here, we demonstrate the value of combining the bone estimate and the residual to produce a "top layer subtracted" (tls) spectrum. We report superior prediction of two standard bone metrics (volumetric bone mineralization density and maximum torque) using partial least squares regression models based upon tls spectra rather than SOLD bone estimates, implying that the spectral residuals contain useful information. Simulations reinforce experimental in vivo findings. This chemometric approach, which we denote as SOLD/TLS, could have broad applicability in situations where comprehensive spectral libraries are difficult to acquire.