Nanoscale imaging of major and minor ampullate silk from the orb-web spider Nephila Madagascariensis

Spider silk fibres have unique mechanical properties due to their hierarchical structure and the nanoscale organization of their proteins. Novel imaging techniques reveal new insights into the macro- and nanoscopic structure of Major (MAS) and Minor (MiS) Ampullate silk fibres from pristine samples of the orb-web spider Nephila Madagascariensis. Untreated threads were imaged using Coherent Anti-Stokes Raman Scattering and Confocal Microscopy, which revealed an outer lipid layer surrounding an autofluorescent protein core, that is divided into two layers in both fibre types. Helium ion imaging shows the inner fibrils without chemical or mechanical modifications. The fibrils are arranged parallel to the long axis of the fibres with typical spacing between fibrils of 230 nm ± 22 nm in the MAS fibres and 99 nm ± 24 nm in the MiS fibres. Confocal Reflection Fluorescence Depletion (CRFD) microscopy imaged these nano-fibrils through the whole fibre and showed diameters of 145 nm ± 18 nm and 116 nm ± 12 nm for MAS and MiS, respectively. The combined data from HIM and CRFD suggests that the silk fibres consist of multiple nanoscale parallel protein fibrils with crystalline cores oriented along the fibre axes, surrounded by areas with less scattering and more amorphous protein structures.