Using hybrid atomic force microscopy and infrared spectroscopy (AFM-IR) to identify chemical components of the hair medulla on the nanoscale.

Atomic force microscopy integrated with infrared spectroscopy (AFM-IR) has been used to topographically and chemically examine the medulla of human hair fibres with nanometre scale lateral resolution. The mapping of cross-sections of the medulla showed two distinct structural components which were subsequently characterised spectroscopically. One of these components was shown to be closely similar to cortical cell species, consistent with the fibrillar structures found in previous electron microscope (EM) investigations. The other component showed large chemical differences from cortical cells and was assigned to globular vacuole species, also confirming EM observations. Further characterisation of the two components was achieved through spectral deconvolution of the protein Amide-I and -II bands. This showed that the vacuoles have a greater proportion of the most thermodynamically stable conformation, namely the antiparallel ß-sheet structures. This chimes with the observed lower cysteine concentration, indicating a lower proportion of restrictive disulphide cross-link bonding. Furthermore, the large α-helix presence within the vacuoles points to a loss of matrix-like material as well as significant intermolecular stabilisation of the protein structures. By analysing the carbonyl stretching region, it was established that the fibrillar, cortical cell-like components showed considerable stabilisation from H-bonding interactions, similar to the cortex, involving amino acid side chains whereas, in contrast, the vacuoles were found to only be stabilised significantly by structural lipids.

Infrared Nanospectroscopy of Air-Sensitive Biological Substrates Protected by Thin Hydrogel Films.

The air sensitivity of many substrates, and specifically biosurfaces, presents an experimental challenge for their analysis by vibrational spectroscopy and, in particular, infrared microscopy on a nanometer scale. The recent development of atomic-force-microscopy-based infrared spectroscopy (AFM-IR), which circumvents the Abbe diffraction limit, allows nanoscale chemical characterization of surfaces. Additionally, this technique has been shown to work for thin films under aqueous environments but is limited to substrates up to 10 nm thick, thus ruling out application to many biological surfaces. To circumvent this restriction, we have utilized hydrogels to cover such surfaces and maintain a more physiologically representative environment for biological substrates. We show that it is feasible to use AFM-IR to chemically characterize this type of substrate buried under a thin hydrogel film. Specifically, this work describes the AFM-IR spectra of red blood cells under polyvinyl alcohol hydrogels.

Nanoscale Molecular Characterization of Hair Cuticle Cells Using Integrated Atomic Force Microscopy-Infrared Laser Spectroscopy.

The hair cuticle provides significant protection from external sources, as well as giving rise to many of its bulk properties, e.g., friction, shine, etc. that are important in many industries. In this work, atomic force microscopy-infrared spectroscopy (AFM-IR) has been used to investigate the nanometer-scale topography and chemical structure of human hair cuticles in two spectral regions. AFM-IR combines atomic force microscopy with a tunable infrared laser and circumvents the diffraction limit that has impaired traditional infrared spectroscopy, facilitating surface-selective spectroscopy at ultra-spatial resolution. This high resolution was exploited to probe the protein secondary structures and lipid content, as well as specific amino acid residues, e.g., cystine, within individual cuticle cells. Characterization across the top of individual cells showed large inhomogeneity in protein and lipid contributions that suggested significant changes to physical properties on approaching the hair edge. Additionally, the exposed layered sub-structure of individual cuticle cells allowed their chemical compositions to be assessed. The variation of protein, lipid, and cystine composition in the observed layers, as well as the measured dimensions of each, correspond closely to that of the epicuticle, A-layer, exocuticle, and endocuticle layers of the cuticle cell sub-structure, confirming previous findings, and demonstrate the potential of AFM-IR for nanoscale chemical characterization within biological substrates.

Spectral Analysis and Deconvolution of the Amide I Band of Proteins Presenting with High-Frequency Noise and Baseline Shifts.

The challenge of deriving quantitative information from the infrared spectra of proteins arises from the large number of secondary structures and amino acid side-chain functional groups that all contribute to the spectral intensity, such as within the amide I band (1600-1700 cm-1). The band is invariably heavily convoluted from overlapping spectral features, thereby making interpretation difficult such that deconvolution is usually required. This work critically examines the methods available to deconvolute the spectra and assesses the commonly used methods and algorithms applied to vibrational spectra for smoothing and peak identification. We show that unless their spectra have very high signal-to-noise ratios, quantitative analysis to decipher protein constituents is not feasible. The advantages and disadvantages of spectral smoothing using adjacent averaging, the Savitzky-Golay filter and the fast Fourier transform filter are examined in detail. The use of derivative spectra to identify peaks is described with particular reference to the influence and reduction of interfering water bands in the amide I region. The reliability of band narrowing techniques such as second-derivative analysis or Fourier deconvolution that lead to the identification of the contributing protein peaks is investigated. Both methods are shown to be limited in their capacity to resolve features with very similar frequencies. Additionally, the presence of narrow bands arising from high-frequency noise whether from atmospheric water vapor, acoustic vibrations, or electrical interference results in both methods becoming increasingly unusable as narrow bands are preferentially enhanced at the expense of broad ones such as the amide I bands. An optimal strategy is critically developed to allow accurate determination and quantification of protein constituents and their conformations. Additionally, quantitative methods are proposed to account for baseline shifts, which would otherwise introduce significant errors in similarity indices.

A structural and temporal study of the surfactants behenyltrimethylammonium methosulfate and behenyltrimethylammonium chloride adsorbed at air/water and air/glass interfaces using sum frequency generation spectroscopy.

Molecular scale information about the structure of surfactants at interfaces underlies their application in consumer products. In this study the non-linear optical technique of Sum Frequency Generation (SFG) vibrational spectroscopy has been used to investigate the structure and temporal behaviour of two cationic surfactants used frequently in hair conditioners. SFG spectra of films of behenyltrimethylammonium methosulfate (BTMS) and behenyltrimethylammonium chloride (BTAC) were recorded at the air/water interface and on glass slides following Langmuir Blodgett (LB) deposition. The assignment of the BTMS and BTAC spectral features (resonances) to the CH stretching modes of the surfactants was consolidated by comparison with the SFG spectrum of deuterated cetyltrimethylammonium bromide (d-CTAB) and by recording spectra on D2O as well as on water. The CH resonances arise from the methylene and methyl groups of the tail and head-groups of the surfactants. A slow collapse mechanism was observed following film compression of both BTAC and BTMS. The change in molecular structure of the films undergoing this slow collapse was followed by recording sequential SFG spectra in the CH region, and by monitoring the SFG intensity at specific wavenumbers over time. Additionally, LB deposition onto glass was used to capture the state of the film during the slow collapse, and these SFG spectra showed close similarity to the corresponding spectra on water. Complementary Atomic Force Microscopy (AFM) was used to elucidate the layering of the compressed and relaxed films deposited onto mica by LB deposition.

Mechanistic Insights into the Challenges of Cycling a Nonaqueous Na-O2 Battery.

Superoxide-based nonaqueous metal-oxygen batteries have received considerable research attention as they exhibit high energy densities and round-trip efficiencies. The cycling performance, however, is still poor. Here we study the cycling characteristic of a Na-O2 battery using solid-state nuclear magnetic resonance, Raman spectroscopy, and scanning electron microscopy. We find that the poor cycling performance is primarily caused by the considerable side reactions stemming from the chemical aggressiveness of NaO2 as both a solid-phase and dissolved species in the electrolyte. The side reaction products cover electrode surfaces and hinder electron transfer across the electrode-electrolyte interface, being a major reason for cell failure. In addition, the available electrode surface and porosity change considerably during cell discharging and charging, affecting the diffusion of soluble species (superoxide and water) and resulting in inhomogeneous reactions across the electrode. This study provides insights into the challenges associated with achieving long-lived superoxide-based metal-O2 batteries.