Polarization-dependent FTIR micro-spectroscopy reveals keratin orientation in longitudinal and transversal hair...

Hair keratin forms fibrils that are oriented along the hair fiber axis. FTIR spectroscopy can be made sensitive to the orientation of molecules in a sample with the use of polarized IR radiation. Although FTIR microspectroscopy was extensively used to study the chemical composition of hairs, it was never extensively used to study the orientation of keratin fibrils in hair sections. We report a methodological study establishing the sensitivity of polarized μFTIR with globar source, Synchrotron Radiation Source and IR Quantum Cascade Laser source to keratin orientation in transversal and longitudinal hair sections, preserving their natural orientation. First, we used a combination of a globar source and an infrared polarizer to study keratin orientation in longitudinal hair sections by rotating the IR polarization parallel and perpendicular to the hair axis. The infrared spectra of longitudinal hair sections under parallel and perpendicular irradiation exhibited major changes in the amide I/amide II ratio while the amide III band exhibited very little polarization sensitivity. The amide A band at 3280 cm^-1 also exhibited polarization sensitivity. We then tested whether transversal hair sections also exhibited polarization sensitivity and found that they indeed exhibited changes in amide I/amide II peak ratios and changes in NH intensity under different polarization orientations showing in plane orientation of the peptide bonds. Synchrotron FTIR microspectroscopy has been extensively used to study hair composition at diffraction limited spatial resolution and synchrotron radiation is inherently polarized: edge radiation is circularly polarized and bending radiation is linearly polarized. We observed changes in amide I/amide II peak ratios in both longitudinal and transversal hair sections measured at two perpendicular angles showing that measurements using naturally linearly polarized synchrotron radiation (without the use of a polarizer) are sensitive to the keratin orientation. The amide III band was less orientation sensitive and should thus be privileged for quantitative measurements when using synchrotron radiation. In the hair medulla spectra, no changes were observed under different orientations showing a lack of significant orientation in some medullas. Infrared Laser Imaging Microscopy spectra were sensitive to the fiber orientation showing that QCL polarization can reveal linear dichroism.