Mapping Nanostructural Variations in Silk by Secondary Electron Hyperspectral Imaging

Abstract

characterization tool to reveal and visualize nanostructural variations across micron-scale spatial dimensions. We report that despite similarity in overall ordered fraction, there are distinct differences in the nanoscale order/ disorder maps of natural silk fibers from both Bombyx mori and Antheraea mylitta.The structural hierarchy of silkworm silks surrounds a sericin glue that binds two microscopic fibroin brins (≈15 µm), [3] which are comprised of ≈200 nm microfibers and nanofibrils [8] and, finally, nanoscale phases, which can be ordered or disordered, [4] as shown in the schematic diagram in Figure 1a.Using low-voltage standard SEM of a cryo-snapped and plasma-exposed silk fibers of B. mori silk and A. mylitta silk, it is possible to visualize bright nanostructures (Figure 1a) due to topographical contrast, but such contrast is problematic for accurate nanoscale dimension measurements due to a feature size-and shape-dependent edge effect. [9] Furthermore, the topography can be caused by different mechanisms, and it is therefore prone to artefacts introduced by sample preparation. Nevertheless, we observe that the average area fraction of these nanostructures is similar in both silks, however in A. mylitta silk, the round nanostructures seem smaller, denser, and interconnected.In order to determine the nature of these bright nanostructures and their dimensions, we applied SEHI. SEHI exploits the distinctiveness of secondary electron (SE) signals in carbonbased material [10] for hyperspectral imaging (HI) and has the advantage of being able to avoid the confounding influence of topology, which beleaguers standard SEM (see Section S6 in the Supporting Information). The concept of HI is well established in vibrational spectroscopies, [11a] where images are formed from several different energy regions, and based on distinctive peaks in the spectrum. This is demonstrated in the schematic in Figure 1d. Here we collect SE spectra from the high-density nanoscale regions, as established by comparison of backscattered electron density maps [12] (Section S1, Supporting Information), which we find correlate with the bright features in the standard SEM images in Figure 1a. This allows us to investigate which peaks in the SE spectra are related to high density (Section S3, Supporting Information), and thus to high order (Section S1, Supporting Information). We then apply the HI concept to quantitatively map the different phases in silks by imaging with an energy window of 3.9 ± 0.3 eV which was specifically selected to map high-order regions and is free from topographical artefacts Nanostructures underpin the excellent properties of silk. Although the bulk nanocomposition of silks is well studied, direct evidence of the spatial variation of nanocrystalline (ordered) and amorphous (disordered) structures remains elusive. Here, secondary electron hyperspectral imaging can be exploited for direct imaging of hierarchical structures in carbon-based materials, which cannot be revealed by any other standard characterizat...

Revealing Spider Silk's 3D Nanostructure Through Low Temperature Plasma Etching and Advanced Low-Voltage SEM

“…The nanostructures evident in Figure 1 inset are most likely a result of the presence of β-sheet rich and ordered nano-domains which etch at a slower rate compared to the disordered amorphous phases. Their appearance is consistent with structures observed in dragline silk by SEM after the shell has been washed away by phosphate buffered saline (Augsten et al, 2000) and are similar to those observed in silkworm silk (Wan et al, 2017).…”

“…In this study a controlled variation of reeling speed is utilized to produce silk of varying mechanical and structural properties. To elucidate the differences in structure, the fibers are analyzed unstained and uncoated in the secondary electron microscope (SEM) by Secondary Electron Hyperspectral Imaging (Wan et al, 2017) and structures are revealed by low temperature plasma etching. The ultimate goal is to spatially characterize the 3D distribution of nanostructure radially and longitudinally and presenting results which will aid in the parameterization of multiscale mechanical models.…”

Characterizing Cross‐Linking Within Polymeric Biomaterials in the SEM by Secondary Electron Hyperspectral Imaging

“…[2,3] Here we show that the SEHI approach can be also utilized as a novel characterization tool to map cross-link densities in beam sensitive biomaterials. While in a standard SEM an image is formed from all detected SEs that are emitted from a surface regardless of their energy, in SEHI a series of images is collected, each of which is created using SEs from a defined energy band.…”

Mapping Polymer Molecular Order in the SEM with Secondary Electron Hyperspectral Imaging

“…A growing body of recent work has established the modern field of secondary electron (SE) energy spectroscopy . This technique in the scanning electron microscope (SEM) has enabled fresh, exciting insights in to the properties of polymeric, organic, and biological materials by exploiting the relationship between the emitted SE energy distribution and various material properties.…”

“…This technique in the scanning electron microscope (SEM) has enabled fresh, exciting insights in to the properties of polymeric, organic, and biological materials by exploiting the relationship between the emitted SE energy distribution and various material properties. By performing energy‐selective detection of SEs in the SEM, techniques such as secondary electron hyperspectral imaging (SEHI) have been used to form images which can map nanoscale variations in chemistry or molecular ordering …”