A nanoscopic journey through spider silk

The remarkable mechanical properties of spider silk have fueled interest in under-standing its material properties and developing an artificial material with similar traits[1, 2]. To develop artificial spider silk, it is necessary to understand the complex structure of the natural silk fibers, as the properties of spider silk arise from both the nanoscopic organiza-tion of its proteins and the hierarchical structure of the silk. Determining the nanostructures within the protein core is challenging, as the techniques previously used require sample alteration or are limited in resolution[3-6]. In this work, the macro- and nanoscopic struc-ture of pristine dragline (Major Ampullate silk, MAS) and auxiliary spiral (Minor Ampullate silk, MiS) silk fibers from the orb-web weaving spider Nephila madagascariensis are exam-ined. Coherent anti-Stokes Raman scattering- and confocal microcopy revealed an outer lipid rich layer of between 600nm-1μm thick. These lipids surround an auto-fluorescent protein core which was divided into two concentric layers in both fiber types. The nano-scopic protein fibrils within pristine spider silk were imaged, for the first time, using scan-ning helium ion microscopy and confocal Raman microscopy. Fibrils were seen to be ar-ranged parallel to the length of the fiber and were present throughout the protein core. The fibral diameters were between 89-228nm. Combined, our results from these different mi-croscopy techniques, thoroughly characterize the microscopic and nanoscopic structures in these fibers with minimal sample alteration. The knowledge of the silks fibral structure and the methods to measure it presented here will support the development of future superior artificial spider silk.