Polarimetric imaging of collagen in histopathology specimens: an investigation of congo red and picrosirius red-stained placenta and skin

Polarisation microscopy is a label-free technique whose tissue architecture profiling through anisotropy remains underinvestigated. In histology, bright-field microscopy and special stains remain the standard for investigating extracellular matrix (ECM) proteins. However, these approaches are qualitative and observer-dependent. By measuring birefringence parameters, quantitative polarisation microscopy (QPM) could offer a more objective alternative. Using dual photoelastic modulators and full Stokes parameter acquisition, QPM was applied to Congo red and picrosirius red-stained sections of human placenta, normal skin, and keloid scars. From the Stokes vectors (I, Q, U, V), polarisation azimuth, ellipticity, phase retardation, and depolarisation maps were derived, enabling quantitative characterisation of collagen organisation. QPM revealed distinct anisotropic signatures corresponding to ECM microarchitecture. Picrosirius red-stained sections demonstrated a three/four-fold in phase retardation increase compared to Congo red, confirming its collagen-specific birefringent enhancement. In placenta, spatially heterogeneous birefringence highlighted perivascular collagen arrangements. In skin, stronger phase retardation and depolarisation patterns were observed in dermal collagen bundles, with keloid scars showing markedly increased phase retardation (~ 1.4 radians) and depolarisation (~ 0.96), reflecting altered collagen density and disorganisation. QPM provides objective, spatially-resolved tissue anisotropy metrics, overcoming the qualitative limitations of traditional histological techniques. This approach offers a robust framework for assessing collagen remodelling in various pathological contexts.