## 3 Results and Discussion  
Dimensions of crystalline \(\beta\)-sheet fibrils in silk fibers is \(\simeq\)10 nm [15]. SNOM measurements are well suited to measure \(n\) and \(\kappa\) from areas of comparable dimensions [11]. Whether it was possible to determine optical anisotropy due to molecular orientation using a point-like excitation source was the motivation of this study. If the anisotropy of absorbance (dichroism \(\Delta\alpha\propto\Delta\kappa\)) and refractive index (birefringence \(\Delta n\)) could be measured with a point-like source using s-SNOM, this would open the possibility of determining anisotropy and molecular orientation with sub-wavelength resolution at nanoscale. The birefringence originates from the alignment of molecular bonds or secondary ordering and can be detected at different spectral ranges [16]. Molecular alignment of the amide-II at 1512 cm\({}^{-1}\) (C-N) and amide-I \(\beta\)-sheets at 1628 cm\({}^{-1}\) (C=O) was measured from micron-thick longitudinal silk slices [17]. In-plane perpendicular orientation between C=O and C-N bonds was revealed [17] using a longitudinal slice of silk fiber, which facilitated fidelity of measurements due to uniformity of thickness [18]. In this study s-SNOM data was collected in the IR spectral range together with topography map (Figure 2). The mechanical amplitude and phase are related to the stability of the feedback loop and were used as a qualitative error signal, i.e., unexpected changes in the mechanical amplitude correlated with errors in the near-field amplitude. The optical amplitude map is also shown (Figure 2) and is dominated by the reflectivity \(\propto n\) of the surface, which is usually very low for the polymers (emissivity \(\propto\kappa\) is high at the absorption bands). Both, mechanical and optical mapping revealed a clear boundary between the longitudinal (90\({}^{\circ}\)-cut) silk fiber slice and surrounding epoxy (Figure 2). Recently, it was demonstrated that mechanical viscoelastic parameters: the Young's modulus, viscosity coefficient, and retardation time can be determined using the amplitude modulated-frequency modulated (AM-FM) mode of cantilever response [19] and are linked to the topography of the sample via the offset of the tapping mode signal. The amide I and II bands [11] were well recognisable in the nano-FTIR absorbance spectra collected from different single measurements points (Figure 2). Measurements were made along 3 \(\upmu\)m linear scan with a separation of 200 nm between the measurement sites. Clear transition from the silk to epoxy was observed and a half-step can be defined as the upper bound of the resolution (Figure 3). The transverse silk slice (0\({}^{\circ}\)-cut) showed similar topography and mechanical (amplitude and phase) mapping with a slightly larger reflectivity (Figure 4). The same scan along the line showed a distinct transition from silk to epoxy. The dark regions along the boundary were observed with spectral features mixed between silk and epoxy (Figure 4). Since the 100 nm thickness of the slice was only approximately twice larger than the diameter of the fiber, mechanical cracks were expected under sheer forces applied during slicing. Comparison of spectra measured from transverse (0\({}^{\circ}\)) and longitudinal (90\({}^{\circ}\)) slices is shown in Figure 5. Different the amide-II and I bands ratios are directly related to the orientation as we showed by the far-field absorbance [17]. For the longitudinal slice, C=O (amide-I) is radially distributed and most strongly coupled to the \(E_{p}\) of the SNOM needle, while C-N (amide-II) is perpendicular to the SNOM tip, hence, uncoupled to the absorbing dipole. The inset in the reflectivity spectrum (Figure 5) shows mutually perpendicular in-plane orientations of the C=O and C-N amide bands on the longitudinal slice; note, the C=O is radially distributed if observed out-of-plane. This illustration also clarifies that for the transverse slice \(E_{p}\parallel\)(C-N) and perpendicular \(E_{p}\perp\)(C=O).