## 4 Conclusions and Outlook  
In summary, spectral characterisation, lateral mapping of nano-IR absorbance and reflectance with deep sub-wavelength resolution in the IR molecular finger printing window was demonstrated using thin (100 nm) transverse and longitudinal slices of silk. Hyper-spectral mapping across the silk fiber slice was obtained with high accuracy and reproducibility. The possibility of orientation measurements using a SNOM tip is proposed and demonstrated at two angles (two slices of silk fiber) using the amide bands and previously measured far-field IR spectra. Since preparation of thin microtome slices of soft  
Figure 5: SNOM maps of the transverse and longitudinal silk slices together with averaged nano-FTIR absorbance and reflectance spectra. Inset in reflectivity panel shows the molecular orientation of the amide-I,II bands in \(\beta\)-sheet crystalline regions for the longitudinal slice (adopted form ref. [17]). Note, chemical bonding in the \(\beta\)-sheets plane only is shown; the C=O bonds have a radial distribution along the fiber direction and their second order momentum distribution was determined in the Supplementary of ref. [17].  
Figure 6: (**a**) Concept of four-angle absorbance measurements where instead of polarisation, four different orientation micro-tome slices are prepared. In this study we carried out measurements on two 90\({}^{\circ}\) and 0\({}^{\circ}\) longitudinal and transverse slices, respectively. (**b**) Visualisation of the a silk fiber with its internal \(\beta\)-sheet structure. (**c**) Orientation determination of C=O \(\beta\)-sheet absorbers at 1628 cm\({}^{-1}\) using different orientation slices (data markets for the transverse \(\theta=0^{\circ}\) and longitudinal \(\theta=90^{\circ}\) data) microtome slices; \(\theta_{o}\) is the initial orientation. Note, for quantitative determination of orientation, four orientation slices are required.  
bio-materials does not alter their structure [11], this opens the possibility of measuring optical properties from nano-volumes prepared by careful alignment of microtome cuts. One can envisage that using slices of silk fiber at different angles 0\({}^{\circ}\) (transverse), \(\pm\)45\({}^{\circ}\), and 90\({}^{\circ}\) (longitudinal), the four-orientation mapping equivalent to the four-polarisation method [20] can be developed and will be focus of a future study. Measurement of orientation changes in the regions of laser-induced material modification by re-melting [22], phase change and ablation [23] will benefit from the introduced method. Recently, orientation and ordering of phospholipids was demonstrated [24].  
Conceptualization, [PERSON], M.R., S.J.; methodology, M.R., J.M., A.C.; experiments and sample preparation, M.R., R.H., A.R., J.H., J.-L.; data analysis, all the authors; writing--review and editing, all the authors.  
This research was funded by JSPS KAKENHI Grants No. 16K06768, 18H04506 (Japan) and the Australian Research Council Discovery projects DP1901032.  
We are grateful for [PERSON] from Materials Analysis Division of Tokyo Institute of Technology, Ookayama, for his assistance with ultramicroomy. [PERSON] is grateful for sabbatical stays at Tokyo Institute of Technology and Shizuoka University in 2018. We thank [PERSON] and [PERSON] for the critical reading of the manuscript.  
The authors declare no conflict of interest.