A quantitative approach for high-throughput mapping of collagen fibril orientation using polarization-dependent Second Harmonic Generation

• Development and validation of a quantitative polarization-dependent Second Harmonic Generation (qPSHG) framework enabling high-throughput, high-resolution mapping of mineralized collagen fibril (MCF) orientation. • qPSHG at sub-micrometer resolution resolves the lamellar architecture of human osteons. • Fourier analysis of qPSHG orientation maps revealed a ∼6.2 µm periodic modulation in out-of-plane fibril angles, directly capturing the alternating collagen orientations that define the helicoidal lamellar structure of osteons. • Quantitative orientation maps demonstrated the circumferential organization of fibrils within osteons, and the oscillatory rotation of fibril angles across successive lamellae, consistent with established structural models. Bone is a hierarchically structured biological composite whose mechanical properties are largely governed by the three-dimensional orientation and arrangement of mineralized collagen fibrils (MCFs) at the ultrastructural level. Investigating this fibrillar organization is crucial for defining the structure-property relationships that govern bone mechanical behavior across length scales and for understanding bone fragility disorders such as Osteogenesis Imperfecta and Paget’s disease. However, existing methods for assessing spatial fibril orientation often require complex sample preparation and do not scale to high-resolution mapping over large areas. Here, we propose a quantitative polarization-dependent Second Harmonic Generation (qPSHG) approach for high-throughput, high-resolution assessment of three-dimensional collagen fibril orientation. The method relies on theoretical calculations of polarization-dependent SHG signal as a function of the orientation of the second-order nonlinear susceptibility tensor, enabling quantitative extraction of fibril orientation through model fitting. This approach is validated using small-angle X-ray scattering (SAXS) measurements on a simplified model system of mineralized collagen fibrils oriented at known angles, where SAXS can provide an independent, ensemble-averaged measure of fibrillar orientation used as a reference. When applied to bone, the method enables sub-micron-resolution mapping of fibrillar orientation, resolving the lamellar arrangement within osteons. By combining high spatial resolution with rapid acquisition, the method is well suited for studies requiring large-scale mapping of bone ultrastructure, holding potential to support investigations of anatomical, age-related, and pathological variations in fibrillar organization. Beyond bone, the qPSHG framework may be adapted to probe the three-dimensional ultrastructure of other collagen-based biological and architectural materials. Bone’s remarkable mechanical properties arise from its hierarchical organization. However, quantitatively mapping the ultrastructural orientation of mineralized collagen fibrils across large bone volumes remains challenging, limiting our ability to link microscale architecture with tissue-level mechanics and disease. This work introduces a quantitative polarization-dependent Second Harmonic Generation (qPSHG) framework that enables rapid, high-resolution, three-dimensional mapping of fibril orientation in bone. Theoretical derivations allow accurate, quantitative extraction of orientation from polarization-dependent SHG data. Applied to human cortical bone, qPSHG reveals the lamellar organization of osteons with sub-micrometer precision and throughput suitable for large-scale analysis. This method provides a powerful new tool for correlating fibrillar architecture with mechanical function and disease-related variation, supporting the development of quantitative biomarkers for bone quality and fragility assessment.