Fig. 4: FIB-SEM 3D-reconstruction of intragranular nanostructure in native thread cuticle.

a FIB-SEM image of osmium stained region of native distal thread showing substructure of the core and cuticle, including granule and matrix in the cuticle and previously described voids in the core. Scale bar = 500 nm. b Higher magnification FIB-SEM image of a single granule. Contrast arises from heavily staining (hs) and lightly staining (ls) regions within a single granule. n.b. contrast is inverted compared to TEM, with elements of higher atomic mass appearing brighter. Scale bar = 200 nm. c 3D reconstruction of FIB-SEM image stack using the contrast of the lightly staining (ls) phase of the granules and the core voids (which are parallel to the fiber axis). d Granule cropped using clipping planes parallel to the one indicated in c revealing internal structure of the ls phase, consisting of bicontinuous flattened layers with a thickness of ~20 nm. The transparent region between layers constitutes the heavily staining (hs) phase from b. Scale bar = 80 nm. e STEM-EDS compositional analysis of native cuticle granule and matrix regions, showing distribution of nitrogen, sulfur, iron, and vanadium in the region in the STEM-HAADF image on the left. Scale bar = 100 nm.