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 ${\mathrm{CH}}_2$ mode measured via the XXY tensor element at $2860{\mathrm{cm}}^{-1}$ 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.