Fast determination of three-dimensional fibril orientation of type-I collagen via macroscopic chirality

Polarization-resolved second harmonic generation (SHG) microscopy is appealing for studying structural proteins and well-organized biophotonic nanostructures, due to its highly sensitized structural specificity. In recent years, it has been used to investigate the chiroptical effect, particularly SHG circular dichroism (SHG-CD) in biological tissues. Although SHG-CD attributed to macromolecular structures has been demonstrated, the corresponding quantitative analysis and interpretation on how SHG correlates with second-order susceptibility χ⁽²⁾ under circularly polarized excitations remains unclear. In this study, we demonstrate a method based on macroscopic chirality to elucidate the correlation between SHG-CD and the orientation angle of the molecular structure. By exploiting this approach, three-dimensional (3D) molecular orientation of type-I collagen is revealed with only two cross polarized SHG images (i.e., interactions of left and right circular polarizations) without acquiring an image stack of varying polarization.