Polarization-Resolved Second Harmonic Generation Microscopy of Silk Fibers Is Sensitive to β-Sheet Orientation and Molecular Structure

Spider silk biomaterials have generated significant interest due to their high strength, biocompatibility, and biodegradability. However, the complex multiscale structure of silk fibers creates difficulties in investigating the relationship between the structure and mechanical properties in silks. Previous work has shown that silks at the focus of an ultrafast laser produce a significant second harmonic generation (SHG) signal. This presents an exciting opportunity since polarization-resolved SHG microscopy (PSHG) is a technique that has shown high sensitivity local molecular structure and organization in several biological samples. However, applications of PSHG to silks have been impeded by the lack of a theoretical model relating silk molecular structure and organization to its PSHG response. Here, a theoretical model of PSHG from silk materials is presented, which relates β-sheet organization within silk fibers to experimentally measurable parameters. Based on this, we present evidence that postspin stretching in ethanol induces planar alignment of the β-sheets within recombinant spider silk fibers, and the molecular structure and degree of axial alignment of β-sheets are highly dependent on the level of postspin stretching. Overall, this work demonstrates the significant potential for the application of PSHG to map the local structure of silk fibers, providing opportunities for the investigation of silk-based biomaterials.