Selective Visualization of Type II Collagen Using Sum‐Frequency Generation ( SFG )

Collagen Types I and II share highly conserved triple‐helical backbones and similar CH stretch vibrational spectra, which limits the ability of conventional spectroscopic or second‐harmonic generation methods to unambiguously distinguish between them in native and engineered matrices. By combining polarization‐resolved sum‐frequency generation (SFG) measurements with tensor‐based simulations of the CH stretch response, this work identifies collagen's asymmetric mode measured via the XXY tensor element at as a robust optical marker that exhibits distinct spatial symmetries for collagen Type I and Type II. In rat auricular cartilage, analysis of the polarization‐resolved SFG signatures combined with vertex component analysis reveals pocket‐like domains of differently oriented collagen Type II fibrils rather than a uniformly aligned network. These findings establish polarization‐resolved SFG microscopy as a structurally specific tool for mapping collagen Type II architecture and label‐free discrimination of collagen Types I and II.