Objective

The use of conventional microscopy and vibrational spectroscopy in the optical region to investigate the chemical nature of hair fibres on a nanometre scale is frustrated by the diffraction limit of light, prohibiting the spectral elucidation of nanoscale sub-structures that contribute to the bulk properties of hair. The aim of this work is to overcome this limitation and gain unprecedented chemical resolution of cortical cell nanostructure of hair.

Methods

The hybrid technique of AFM-IR, combining atomic force microscopy with an IR laser, circumvents the diffraction limit of light and achieves nanoscale chemical resolution down to the AFM tip radius. In this work, AFM-IR was employed on ultra-thin microtomed cross-sections of human hair fibres to spectrally distinguish and characterise the specific protein structures and environments within the nanoscale components of cortical cells.

Results

At first, a topographical and chemical distinction between the macrofibrils and the surrounding intermacrofibillar matrix was achieved based on 2.5x2.5μm maps of cortical cell cross-sections. It was found that the intermacrofibrillar matrix has a large protein content and specific cysteine-related residues, whereas the macrofibrils showed bigger contributions from aliphatic amino acid residues and acidic-/ester-containing species (e.g. lipids). Localised spectra recorded at a spatial resolution of the order of the AFM tip radius enabled the chemical composition of each region to be determined following deconvolution of the Amide-I and Amide-II bands. This provided specific evidence for a greater proportion of α-helices in the macrofibrils and correspondingly larger contributions of β-sheet secondary structures in the intermacrofibrillar matrix, as inferred in earlier studies. Analysis of the parallel and antiparallel β-sheet structures, and of selected dominant amino acid residues, yielded further novel composition and conformation results for both regions.

Conclusion

In this work, we overcome the diffraction limit of light using atomic force microscopy integrated with IR laser spectroscopy (AFM-IR) to characterise sub-micron features of the hair cortex at ultra-high spatial resolution. The resulting spectral analysis shows clear distinctions in the Amide bands in the macrofibrils and surrounding intermacrofibrillar matrix, yielding novel insight into the molecular structure and intermolecular stabilisation interactions of the constituent proteins within each cortical component.