Label-free imaging of matrix mineralization in alginate-encapsulated bone spheroids using Coherent Raman Scattering microscopy

Three-dimensional (3D) cell cultures, such as spheroids, are increasingly used to perform advanced studies on bone matrix mineralization. However, their full characterization remains challenging. Traditional colorimetric and fluorescent assays using dyes, such as Alizarin Red S (ARS) and calcein, are effective in monolayer cell cultures, but fail to provide reliable information when used in complex 3D cell constructs. In this study, we investigated the application of Coherent Raman Scattering microscopy for label-free, comprehensive characterization of extracellular matrix (ECM) mineralization in alginate-encapsulated bone spheroids. After confirming that traditional staining techniques are unreliable for mineral detection in spheroids, Stimulated Raman Scattering (SRS) microscopy was used to detect phosphate-rich mineral deposits at a Raman shift of 960 cm-1, while Second Harmonic Generation (SHG) microscopy was used in association with SRS to provide complementary information on the deposition and organization of the collagenous matrix. SRS was used to detect lipid-rich regions at a Raman shift of 2857 cm-1 to perform cell localization. SRS imaging revealed the presence of phosphate-rich regions in the spheroids, including the core regions, usually challenging to characterize in intact 3D constructs. Raman spectral scans on SRS-positive regions confirmed the specificity of the phosphate signal. In addition, comparison of SRS and Coherent Anti-Stokes Raman Scattering (CARS) demonstrated the advantage of SRS in terms of reduced background compared to CARS for lipid imaging. Taken together, our results demonstrated that SRS, in combination with SHG, provides a promising and powerful approach to perform label-free, chemically specific characterization of intact 3D bone models.