The effect of extracellular matrix properties on polarized light-based analysis of collagen fiber alignment in soft tissues

Polarimetry has long been used to investigate fiber anisotropy of biological soft tissues, either independently or together with other imaging methods, as it leverages the natural birefringence of collagen. Despite widespread usage, previous studies of soft tissues contain ambiguous interpretations of data gathered from polarized light-based techniques. To date, there has not yet been a systematic assessment of how individual extracellular matrix (ECM) properties influence the polarization of light, which limits the ability to correctly interpret data from these techniques in some applications. To probe the effect of various ECM properties on polarized light, we used a tunable hydrogel system to vary the collagen density, crosslinking density, and absorber concentration. Samples were imaged using quantitative polarized light imaging (QPLI), which uses circularly polarized incident light and a division of focal plane polarimeter. QPLI was performed in both reflectance and transmission modes. The average degree of linear polarization (AVG DoLP; i.e., strength of alignment) and standard deviation of the angle of polarization (STD AoP; i.e., uniformity of alignment) were calculated for each hydrogel. Increasing collagen density resulted in the most pronounced changes, where AVG DoLP and STD AoP increased for reflectance and transmission mode, likely due to the increased concentration of birefringent material. Crosslinking only caused a modest increase on AVG DoLP in transmission mode but decrease in STD AoP in reflectance mode, likely due to the small length scale of the crosslink relative to the fibers. Alteration of transmissivity resulted in changes mainly in reflectance mode, where multiple scattering was more pronounced. Results will help improve data interpretation and experimental control when using polarized light to image biological soft tissues.