Raman spectroscopy based framework for <i>in-vivo</i> assessment of bone composition

Abstract Bone-related disorders, including osteoporosis, lead to decreased bone density and altered microarchitecture, significantly increasing the risk of fragility fracture. There is an urgent need for innovative early detection methods, as conventional techniques like dual-energy x-ray absorptiometry offer limited insights into bone quality, often overlooking critical microstructural changes. In this study, we present a novel dual-wavelength inverse spatially offset Raman spectroscopy (DWiSORS) system specifically designed to enhance the assessment of transcutaneous bone signals. This innovative approach utilises ring illumination to deliver adequate power while minimising the fluorescence background, and dual-wavelength excitation to capture a broader molecular profile (spectral range). We introduce a novel metric, enhancement-to-noise ratio (ENR), which provides a quantitative and robust strategy to identify the optimal offset for accurate assessment at specified depths, addressing a significant limitation in existing in-vivo SORS methodologies. The metric efficacy was validated through in-vivo measurements on ten healthy volunteers, where the ENR was analysed against increasing source-detector offsets, with tissue thickness determined by ultrasound imaging. Notably, optimal signal-to-noise ratio for bone signals were achieved at offsets of 7 or 9 mm, even beneath 10–14 mm of overlaying tissue, reinforcing increasing the offset beyond this threshold does not necessarily enhance bone signals detection. In-vivo measurements demonstrate the technical feasibility and robustness of the proposed DWiSORS framework for depth-resolved bone Raman measurements. This study indicates the potential of Raman spectroscopy to complement conventional imaging techniques and support future approaches for non-invasive assessment and longitudinal monitoring of bone-related disorders.