Investigating Bénard-Marangoni migration at the air-water interface in the time domain using sum frequency generation (SFG) spectroscopy of palmitic acid monolayers.

Sum-frequency generation (SFG) spectroscopy is frequently used to investigate the structure of monolayer films of long-chain fatty acids at the air-water interface. Although labeled a non-invasive technique, introducing intense SFG lasers onto liquid interfaces has the potential to perturb them. In the present work, narrowband picosecond SFG is used to study the structural changes that occur in palmitic acid and per-deuterated palmitic acid monolayers at the air-water interface in response to the high field strengths inherent to SFG spectroscopy. In order to determine structural changes and identify measurement artifacts, the changes in specific resonance intensities were measured in real-time and over a broad range of surface concentrations from films spread onto a stationary Langmuir trough. Using narrowband instead of broadband SFG minimizes the overlap of the incident infrared beam in the lipid C-H stretching region with resonances from the water sub-phase. Nevertheless, narrowband SFG still generates a thermal gradient at the surface, which produces a significant decrease in local concentration in the area of the laser spot caused by Bérnard-Marangoni convection originating in the sub-phase. The decrease in concentration results in an increase in the conformational disorder and a decrease in the tilt angle of lipid tails. Crucially, it is shown that, even at the highest monolayer concentrations, this gives rise to a measurement effect, which manifests itself as a dependence on the spectral acquisition time. This effect should be taken into account when interpreting the structure of monolayer films on liquid surfaces deduced from their SFG spectra.

Chemically characterizing the cortical cell nano-structure of human hair using atomic force microscopy integrated with infrared spectroscopy (AFM-IR).

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 was to overcome this limitation and gain unprecedented chemical resolution of cortical cell nano-structure 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 characterize 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 2.5 × 2.5 µm 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). Localized 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 ß-sheet secondary structures in the intermacrofibrillar matrix, as inferred in earlier studies. Analysis of the parallel and antiparallel ß-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 characterize 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 stabilization interactions of the constituent proteins within each cortical component.

OBJECTIF: L'utilisation de la microscopie conventionnelle et de la spectroscopie vibratoire dans la région optique pour étudier la nature chimique des fibres capillaires à l'échelle nanométrique est limitée par la limite de diffraction de la lumière, interdisant l'élucidation spectrale des sous-structures à l'échelle nanométrique qui contribuent aux propriétés des cheveux en général. L'objectif de ce travail était de surmonter cette limitation et d'obtenir une résolution chimique sans précédent de la nanostructure cellulaire corticale des cheveux. MÉTHODES: La technique hybride de l'AFM-IR, combinant la microscopie à force atomique avec un laser IR, contourne la limite de diffraction de la lumière et permet d'obtenir une résolution chimique à l'échelle nanométrique jusqu'au rayon de l'extrémité de l'AFM. Dans ce travail, l'AFM-IR a été employée sur des coupes transversales microtomes ultrafines de fibres de cheveux humains pour distinguer et caractériser sur le plan spectral les structures et environnements protéiques spécifiques au sein des composants à l'échelle nanométrique des cellules corticales. RÉSULTATS: Tout d'abord, une distinction topographique et chimique entre les macrofibrilles et la matrice intermacrofibrillaire environnante a été obtenue à partir de cartes de 2,5 × 2,5 micromètres des coupes transversales des cellules corticales. Il a été constaté que la matrice intermacrofibrillaire avait une grande teneur en protéines et des résidus spécifiques liés à la cystéine, tandis que les macrofibrilles présentaient des contributions plus importantes provenant de résidus d'acides aminés aliphatiques et d'espèces acides/contenant des esters (p. ex. lipides). Les spectres localisés enregistrés à une résolution spatiale de l'ordre du rayon de l'extrémité AFM ont permis de déterminer la composition chimique de chaque région suite à la déconvolution des bandes Amide-I et Amide-II. Cela a apporté des preuves spécifiques pour une plus grande proportion des hélices alpha des macrofibrilles, de même que des contributions plus importantes des structures secondaires à feuillet bêta dans la matrice intermacrofibrillaire, déduites dans des études antérieures. L'analyse des structures parallèles et antiparallèles des feuillets bêta, et des résidus d'acides aminés dominants sélectionnés a donné des résultats inédits de composition et de conformation pour les deux régions. CONCLUSION: Dans ce travail, nous avons surmonté la limite de diffraction de la lumière en utilisant la microscopie à force atomique intégrée à la spectroscopie laser IR (IR-AFM) pour caractériser les caractéristiques submicroniques du cortex capillaire à une résolution spatiale ultra-élevée. L'analyse spectrale qui en résulte montre des distinctions nettes dans les bandes d'amide dans les macrofibrilles et la matrice intermacrofibrillaire environnante, ce qui apporte un éclairage nouveau sur la structure moléculaire et les interactions de stabilisation intermoléculaire des protéines constitutives dans chaque composant cortical.

Nanoscale adhesion profiling and membrane characterisation in sickle cell disease using hybrid atomic force microscopy-IR spectroscopy.

Sickle cell disease (SCD) presents a significant global health problem. At present there is no effective treatment, with most being supportive for its associated complications such as the vaso-occlusive crises that result from increased cell adhesion. Hypoxic sickle cells have previously shown greater phosphatidylserine (PS) exposure and oxidative damage, as well as being notably "stickier" suggesting that increased cell cohesion and adhesion to the blood vessel endothelium is a possible mechanism for vaso-occlusion. The present work uses the hybrid technique of atomic force microscopy nano-infrared spectroscopy (AFM-IR) to probe changes to the coefficient of friction and C-O IR intensity in SCD on a nanoscale for dried red blood cells (RBCs) fixed under conditions of hypoxia and correlates these observations with adhesive interactions at the membrane. Using functionalised AFM tips, it has been possible to probe adhesive interactions between hydrophilic and hydrophobic moieties exposed at the surface of the dried RBCs fixed under different oxygenation states and for different cell genotypes. The results are consistent with greater PS-exposure and oxidative damage in hypoxic sickle cells, as previously proposed, and also show strong correlation between localised oxidative damage and increased adhesion. A mechanistic explanation involving significant lipid tail disruption as a result of oxidative action, in combination with differing concentrations of externalised PS lipids, is proposed to explain the observed adhesion behaviour of each type of cell.

A time domain study of surfactin penetrating a phospholipid monolayer at the air-water interface investigated using sum frequency generation spectroscopy, infrared reflection absorption spectroscopy, and AFM-nano infrared microscopy.

We have investigated the interaction of surfactin with a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water interface as a function of time, following its injection into the sub-phase, using non-linear Sum Frequency Generation (SFG) vibrational spectroscopy and Infrared Reflection Absorption Spectroscopy (IRRAS). SFG resonances from the phospholipid and from the surfactin were distinguished from each other by using selective deuteration. The surface pressure at the interface was measured concurrently for up to 8 h. After an induction period, the spectra from the lipid diminished and those of surfactin gradually appeared whilst at the same time the surface pressure increased. However, eventually the surfactin signals disappeared and those of the lipid reappeared. Although the SFG spectra of the lipid disappeared at intermediate times, the IRRAS spectra of the lipid were always present at the interface. Variation in the temporal SFG behaviour was investigated as the pH of the sub-phase, the initial surface pressure of the lipid, and the surfactin concentration were changed. Samples of the surface film were transferred onto mica substrates at selected times along the temporal profile and imaged by Atomic Force Microscopy - nano Infrared Spectroscopy (nano-IR). A model is proposed to account for the results from the four different experimental techniques used.

Structure of the Fundamental Lipopeptide Surfactin at the Air/Water Interface Investigated by Sum Frequency Generation Spectroscopy.

The lipopeptide surfactin produced by certain strains of Bacillus subtilis is a powerful biosurfactant possessing potentially useful antimicrobial properties. In order to better understand its surface behavior, we have used surface sensitive sum frequency generation (SFG) vibrational spectroscopy in the C-H and C═O stretching regions to determine its structure at the air/water interface. Using surfactin with the leucine groups of the peptide ring perdeuterated, we have shown that a majority of the SFG signals arise from the 4 leucine residues. We find that surfactin forms a robust film, and that its structure is not affected by the number density at the interface or by pH variation of the subphase. The spectra show that the ring of the molecule lies in the plane of the surface rather than perpendicular to it, with the tail lying above this, also in the plane of the interface.

Orientation of cholesterol in hybrid bilayer membranes calculated from the phases of methyl resonances in sum frequency generation spectra.

The phases of Sum Frequency Generation (SFG) vibrational resonances recorded from thin films on metal surfaces provide information on the orientation and tilt angles of the functional groups of molecules in the film. SFG spectra have been simulated for monolayer films in which the adsorbed molecule has an unequal number of methyl groups oriented in two different directions. The phases, on resonance, of the methyl symmetric (r(+)) and asymmetric (r(-)) resonances are determined as a function of the two methyl group tilt angles and the fraction of groups pointing in that particular direction. The results are first presented as two-dimensional projection plots for the r(+) and r(-) resonances and then combined to show the tilt angles of the methyl groups, and fraction of methyl groups in that orientation, for which both resonances are in phase or out of phase with one another. Mathematical expressions have been calculated to identify the precise boundary conditions for when the phases of the r(+) and r(-) resonances change. The results of these simulations are compared to the phases of the methyl resonances in experimental SFG spectra of d7-cholesterol in a hybrid bilayer membrane.

Structure of mixed phosphatidylethanolamine and cholesterol monolayers in a supported hybrid bilayer membrane studied by sum frequency generation vibrational spectroscopy.

The structure of hybrid bilayer membranes (HBMs) containing either a pure cholesterol or mixed cholesterol/dipalmitoylphosphatidylethanolamine (DPPE) proximal layer adsorbed onto an octadecanethiol (ODT) self-assembled monolayer (SAM) on a gold substrate have been investigated by sum frequency generation (SFG) spectroscopy. The HBMs were formed by the adsorption of either a pure cholesterol or mixed DPPE/cholesterol monolayer from the air/water interface of a Langmuir-Blodgett trough at surface pressures of 1, 20, or 40 mN·m(-1). SFG spectra were also recorded of HBMs where cholesterol was replaced by cholesterol-d(7), in which the terminal isopropyl group of the alkyl chain of cholesterol was isotopically labeled. In order to isolate the contribution to the SFG spectra from the cholesterol in the mixed cholesterol/phospholipid films, DPPE-d was used, in which the alkyl chains of the phospholipid were deuterated. The infrared spectra of solvent-cast cholesterol and cholesterol-d(7) films were recorded to aid with assignment of the SFG spectra of the HBMs. Features corresponding to methyl, methylene, and methine stretches of cholesterol were identified in the SFG spectra. Information on the polar orientation of SFG-active groups was obtained from the phases of the spectral features. The structure of the HBMs showed little dependence on the surface pressure at which they were formed. SFG spectra of HBMs with a mixed cholesterol/DPPE proximal layer were very similar to the spectra of HBMs with a pure cholesterol proximal layer, although the features in the spectra were more intense than anticipated for a film with half the number of cholesterol molecules, indicating that DPPE did have some effect on the orientation of cholesterol molecules in the film.

Line strengths and transition dipole moment of the nu2 fundamental band of the methyl radical.

The line strengths of nine Q-branch lines in the nu(2) fundamental band of the methyl radical in its ground electronic state have been measured by diode laser absorption spectroscopy. The vibration-rotation spectrum of methyl was recorded in a microwave discharge in ditertiary butyl peroxide heavily diluted in argon. The absolute concentration of the radical was determined by measuring its kinetic decay when the discharge was extinguished. The translational, rotational, and vibrational temperatures, also required to relate the line strengths to the transition dipole moment, were determined from relative integrated line intensities and from the Doppler widths of the lines after allowing for instrumental factors. The line strengths of the nine Q-branch lines were used to derive a more accurate value of the transition dipole moment of this band, mu(2)=0.215(25) D. Improved accuracy over earlier measurements of mu(2) (derived from line strengths of single lines) was obtained by integrating over the complete line profile instead of measuring the peak absorption and assuming a Doppler linewidth to deduce the concentration. In addition, a more precise value for the rate constant for methyl radical recombination than available earlier was employed. The new value of mu(2) is in very good agreement with high-quality ab initio calculations. Furthermore, the ratio of the transition dipole moments of the nu(2) and nu(3) fundamental bands in the gas phase is now in highly satisfactory agreement with the ratio determined for the condensed phase.

Diode laser spectroscopy of the fundamental bands of 12C14N, 13C14N, 12C15N, 13C15N free radicals in the ground 2 Sigma+ electronic state.

Rotationally resolved spectra of the fundamental band of the CN free radical in four isotopic forms have been measured using tunable diode laser absorption spectroscopy. The source of the radical was a microwave discharge in a mixture of isotopically selected methane and nitrogen diluted with argon. The lines were measured to an accuracy of 5 x 10(-4) cm(-1) and fitted to the formula for the vibration rotation spectrum of a diatomic molecule, including quartic distortion constants. The band origins of each of the isotopomers from the five parameter fits were found to be 12C14N: 2042.42115(38) cm(-1), 13C14N: 2000.08479(23) cm(-1), 12C15N: 2011.25594(25) cm(-1), 13C15N: 1968.22093(33) cm(-1) with one standard deviation from the fit given in parenthesis. Some of the lines showed a resolved splitting due to the spin rotation interaction. This was averaged for fitting purposes. The average equilibrium internuclear distance derived from the upsilon = 0 and 1 rotational constants of the four isotopomers is 1.171800(6) A which is in good agreement with the value determined from microwave spectroscopy.

Trace gas absorption spectroscopy using laser difference-frequency spectrometer for environmental application.

A widely tunable infrared spectrometer based on difference frequency generation (DFG) has been developed for organic trace gas detection by laser absorption spectroscopy. On-line measurements of concentration of various hydrocarbons, such as acetylene, benzene, and ethylene, were investigated using high-resolution DFG trace gas spectroscopy for highly sensitive detection.