Molecular alignment underpins optical, mechanical, and thermal properties ofmaterials, however, its direct measurement from volumes with micrometerdimensions is not accessible, especially, for structurally complexbio-materials. How the molecular alignment is linked to extraordinaryproperties of silk and its amorphous-crystalline composition has to be accessedby a direct measurement from a single silk fiber. Here, we show orientationmapping of the internal silk fiber structure via polarisation-dependent IRabsorbance at high spatial resolution of 4.2 micrometers and 1.9 micrometers ina hyper-spectral IR imaging by attenuated total reflection using synchrotronradiation in the spectral fingerprint region around 6 micrometers wavelength.Free-standing longitudinal micro-slices of silk fibers, thinner than the fibercross section, were prepared by microtome for the four polarisation method todirectly measure the orientational sensitivity of absorbance in the molecularfingerprint spectral window of the amide bands of b-sheets and amorphouspolypeptides of silk. Flat lateral micro-slices of silk eliminates shaperelated artefact in determination of absorbance anisotropy and order parametersof the amide bands.
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