Background: Nanoscale composition of silk defining its unique properties via a hierarchical structural anisotropy has to be analysed at the highest spatial resolution of tens-of-nanometers corresponding to the size of fibrils made of b-sheets, which are the crystalline building blocks of silk. Results: Nanoscale optical and structural properties of silk have been measured from 100-nm thick longitudinal slices of silk fibers with ~10 nm resolution, the highest so far. Optical sub-wavelength resolution in hyperspectral mapping of absorbance and molecular orientation were carried out for comparison at IR wavelengths 2-10 micrometers using synchrotron radiation. Conclusion: Reliable distinction of transmission changes by only 1-2% due to anisotropy of amide bands was obtained from nano-thin slices of silk.