Quantitative Imaging of Collagen Fibrils via Correlated Far-Field and Near-Field Optical Techniques

Collagen fibrils are essential for the structural and functional integrity of biological tissues, and their imaging is key to understanding tissue health and pathology. We present a multimodal workflow combining far-field polarization-resolved second harmonic generation (PSHG) microscopy with scattering-type scanning near-field optical microscopy (s-SNOM) for comprehensive collagen characterization. PSHG provides diffraction-limited information on fibril anisotropy and helical pitch angle, while s-SNOM, paired with atomic force microscopy, yields nanoscale optical properties such as refractive index and extinction coefficient alongside topographic context. Using type I collagen fibrils from bovine tendons, we examine structural and optical changes induced by noncollagenous matrix elements, laser-induced denaturation, oxidative stress, and thermal exposure. These experiments demonstrate the versatility of our approach in probing subtle collagen structural details and reveal complementary insights into collagen degradation beyond the reach of conventional far-field techniques.