  Molecular alignment underpins optical, mechanical, and thermal properties of
materials, however, its direct measurement from volumes with micrometer
dimensions is not accessible, especially, for structurally complex
bio-materials. How the molecular alignment is linked to extraordinary
properties of silk and its amorphous-crystalline composition has to be accessed
by a direct measurement from a single silk fiber. Here, we show orientation
mapping of the internal silk fiber structure via polarisation-dependent IR
absorbance at high spatial resolution of 4.2 micrometers and 1.9 micrometers in
a hyper-spectral IR imaging by attenuated total reflection using synchrotron
radiation in the spectral fingerprint region around 6 micrometers wavelength.
Free-standing longitudinal micro-slices of silk fibers, thinner than the fiber
cross section, were prepared by microtome for the four polarisation method to
directly measure the orientational sensitivity of absorbance in the molecular
fingerprint spectral window of the amide bands of b-sheets and amorphous
polypeptides of silk. Flat lateral micro-slices of silk eliminates shape
related artefact in determination of absorbance anisotropy and order parameters
of the amide bands.
