Label-free volumetric refractive-index imaging of fibrillar collagen architecture, assembly, and cell-associated remodeling

Fibrillar collagen architecture regulates tissue mechanics and cell-matrix interactions, but following how collagen-rich matrices assemble and remodel in three dimensions requires non-destructive measurements that can link structure to matrix-associated mass without exogenous labels. Here we present holotomography (HT) as a label-free platform for volumetric imaging and quantitative profiling of reconstituted fibrillar collagen. Volumetric RI tomograms resolved fibrillar collagen networks without exogenous labels, and co-registration with second-harmonic generation (SHG) microscopy supported the correspondence between HT-resolved RI structures and SHG-positive fibrillar collagen. Curvelet-transform-based segmentation of representative axial sections converted the tomograms into fiber-level descriptors, including morphology, orientation and RI-derived fiber-associated dry mass. At the matched 0.8 mg/mL condition, HT resolved distinct network organization, with type I forming thicker, more sparsely distributed fibers and type III a finer, denser network, while mean fiber-associated RI remained comparable between subtypes. Across the concentration series, type I showed stronger increases in fiber width and RI-derived mass, whereas type III remained comparatively fine. Time-lapse HT further captured RI dynamics during type I fibrillogenesis and visualized local cell-matrix reorganization in HT1080 cell-embedded gels, capturing cells and surrounding matrix in a single label-free measurement. Together, these results position HT as a complementary label-free approach for volumetric visualization and RI-based quantification of collagen-rich extracellular matrices.