Abstract.SignificanceDivision-of-focal-plane Stokes polarimetry is emerging as a powerful tool for the microstructural characterization of soft tissues. How individual extracellular matrix (ECM) properties influence polarimetric signals in reflectance or transmission modes of quantitative polarized light imaging (QPLI) is not well understood.AimWe aimed to investigate how ECM properties affect outcomes obtained from division-of-focal-plane polarimetric imaging in reflectance or transmission modes.ApproachTunable collagen gel phantoms were used to modulate ECM properties of anisotropy, collagen density, crosslinking, and absorber density; the effects of degree of linear polarization (DoLP) and angle of polarization (AoP) on polarimetry outcomes were assessed. A model biological tissue (i.e., bovine tendon) was similarly imaged and evaluated using both reflectance and transmission modes.ResultsReflectance QPLI resulted in decreased DoLP compared with transmission mode. A 90 deg shift in AoP was observed between modes but yielded similar spatial patterns. Collagen density had the largest effect on outcomes besides anisotropy in both imaging modes.ConclusionsBoth imaging modes were sufficiently sensitive to detect structural anisotropy differences in gels of varying fiber alignment. Conclusions drawn from phantom experiments should carry over when interpreting data from more complex tissues and can help provide context for interpretation of other Stokes polarimetry data.