Second harmonic generation of myofibrils exhibits a polarization-resolved “gradient” effect

Polarization-resolved second harmonic generation microscopy (PSHG) was used to investigate the ultrastructure of individual myofibrils and collagen fibrils to visualize the <i>ρ</i> parameter. A gradient in the <i>ρ</i> values was experimentally observed orthogonal to the myofibrils, and numerical focal volume simulations were performed, indicating myosin SHG has an imaginary chiral component, similar to collagen fibrils. Both types of fibrils were re-imaged after a 180° rotation to investigate the physical mechanism behind the gradient effect. A change in the gradient direction was often observed in myofibrils, but less so in collagen, where it was more variable. PSHG is often used to discriminate muscle and collagen diseases, and chiral SHG contributions, which are typically ignored, should be considered when interpreting PSHG data.