Polarization-resolved super-resolution second-harmonic generation imaging based on multifocal structured illumination microscopy.

Polarization-resolved second-harmonic generation (PSHG) microscopy is widely used in investigating the structural and morphological alterations of collagen. However, the resolution of second-harmonic generation (SHG) imaging remains constrained by optical diffraction, resulting in the polarization extraction of collagen characteristics from the average properties of collagen fibers. In this study, multifocal structured illumination microscopy (MSIM) was combined with PSHG to achieve polarization-resolved super-resolution imaging of second-harmonic generation signals. For the first time to our knowledge, periodic structures with an average pitch of 277 nm were observed in mouse tail tendons using optical microscopy, and the orientation angle of fibrils within each period was found to exhibit an alternating arrangement along the axis in a regular pattern.