TMAO perturbs intermolecular vibrational motions of water revealed by low-frequency modes.

Trimethylamine N-oxide (TMAO) as a representative natural osmolyte has received much attention because of its unique properties, including enhancement of hydrogen bonding networks in liquid water and stabilization of three-dimensional structures of proteins in living organisms. As a hydrogen bond maker and/or a protein stabilizer, its hydrated structures and orientation dynamics in aqueous solutions have been investigated by various spectroscopic methods. Particularly, distinct from other natural osmolytes, it has been found that TMAO molecules form complexes with water molecules even at low concentrations, showing extraordinarily long lifetimes and much larger effective dipole moments. In this study, we demonstrated that collective motions of water molecules are closely correlated to TMAO molecules, as revealed by the changes of the librational modes observed in hyper-Raman (HR) spectra in the low-frequency region (<1000 cm-1) for the first time. Based on HR spectra of the TMAO solutions at submolar concentrations, we observed that the librational bands originating from water apparently upshift (∼15 cm-1) upon the addition of TMAO molecules. Compared to the OH stretching band of water showing a negligible downshift (<5 cm-1), the librational bands of water are more sensitive to reflect changes in the hydrogen bonding networks in the TMAO solutions, suggesting formation of transient TMAO-water complexes plays an essential role toward surrounding water molecules in perturbing their librational motions. We expect to provide a supplementary approach to understand that water molecules in TMAO aqueous solutions are strongly affected by TMAO molecules, different from other osmolytes.

N=8 Armchair Graphene Nanoribbons: Solution Synthesis and High Charge Carrier Mobility.

Structurally defined graphene nanoribbons (GNRs) have emerged as promising candidates for nanoelectronic devices. Low band gap (<1 eV) GNRs are particularly important when considering the Schottky barrier in device performance. Here, we demonstrate the first solution synthesis of 8-AGNRs through a carefully designed arylated polynaphthalene precursor. The efficiency of the oxidative cyclodehydrogenation of the tailor-made polymer precursor into 8-AGNRs was validated by FT-IR, Raman, and UV/Vis-near-infrared (NIR) absorption spectroscopy, and further supported by the synthesis of naphtho[1,2,3,4-ghi]perylene derivatives (1 and 2) as subunits of 8-AGNR, with a width of 0.86 nm as suggested by the X-ray single crystal analysis. Low-temperature scanning tunneling microscopy (STM) and solid-state NMR analyses provided further structural support for 8-AGNR. The resulting 8-AGNR exhibited a remarkable NIR absorption extending up to ∼2400 nm, corresponding to an optical band gap as low as ∼0.52 eV. Moreover, optical-pump TeraHertz-probe spectroscopy revealed charge-carrier mobility in the dc limit of ∼270 cm2  V-1 s-1 for the 8-AGNR.

Hyper-Raman spectroscopy of benzene and pyridine revisited.

Hyper-Raman (HR) spectra of benzene-h6, benzene-d6, and pyridine in the liquid phase excited at 1064 nm were measured by a picosecond laser with a high repetition rate. Although benzene and pyridine are important aromatic molecules, the qualities of the HR spectra previously reported were not high enough to be compared with those of IR and Raman spectroscopy. Our HR spectroscopic system significantly improves sensitivity that enables the detection of HR bands of benzene and pyridine not observed before. In addition to band assignments, we interpret HR bands of benzene based on the vibronic coupling theory of (pre-) resonance hyper-Raman scattering. Depolarization ratios of HR bands of benzene and pyridine, obtained from polarized-HR measurements, are first examined from a theoretical point of view of HR spectroscopy. Moreover, we evaluate quantum chemical calculations for HR spectra by comparing experimental and computational spectra. We show that the frequency-dependent polarizability and hyperpolarizability calculations using time-dependent density functional theory well reproduce the HR experiments for bulk aromatic compounds.

Hyper-Raman spectroscopy of polar liquids excited at 1064 nm: Acetone, acetonitrile, chloroform, and dimethyl sulfoxide.

Hyper-Raman (HR) spectra of polar liquids are reported. Acetone, acetonitrile, chloroform, and dimethyl sulfoxide in the liquid phase were measured by using a picosecond laser whose wavelength is 1064 nm and repetition rate is 200 kHz. HR spectra with a high signal to noise ratio were obtained without the surface enhancement or the electronic resonance effect. Due to the improvement of the sensitivity, many vibrational bands were first observed in HR spectroscopy. The peak frequencies, relative intensities, band assignments, including symmetry species, and depolarization ratios are examined. All IR active vibrational modes well separated were, indeed, observed in HR spectra following the selection rule, whereas HR spectra show not only similarity but also difference in relative signal intensities compared with IR spectra. This work demonstrates the possibility of HR spectra in the liquid phase and suggests further research on molecular structures by HR spectroscopy.

Bulk-or-interface assignment of heterodyne-detected chiral vibrational sum frequency generation signal by its polarization dependence.

Polarization dependence of heterodyne-detected chiral vibrational sum frequency generation (VSFG) was examined for thin films of polylactic acids and neat limonene liquid far from electronic resonance. The enantiomers of polylactic acid films on silica substrates were successfully distinguished, and their chiral VSFG signals were ascribed not to bulk but to the interfaces by comparing chiral signals observed in reflection in the S-polarized VSFG, P-polarized visible, and P-polarized infrared and P-polarized VSFG, S-polarized visible, and P-polarized infrared polarization combinations with theoretical model calculations. In the same way, the chiral VSFG signal of neat limonene was assigned to bulk, which is consistent with the previous assignment. The method employed for assigning the source of chiral signals to the bulk or the interface may be useful for organic films on substrates with low refractive indices and thick samples.

Molecular conformation of DPPC phospholipid Langmuir and Langmuir-Blodgett monolayers studied by heterodyne-detected vibrational sum frequency generation spectroscopy.

Heterodyne-detected (phase-sensitive) vibrational sum frequency generation spectroscopy was used to investigate molecular structures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers on water (Langmuir monolayer) and monolayers on a fused silica substrate (Langmuir-Blodgett [LB] monolayer). The spectral features in the CH stretching region depended on the phase of the Langmuir monolayer, which was controlled by the molecular area on water. From the spectral changes, the molecular structure of the monolayer in each phase was deduced at the molecular level. We discovered that when we compared Langmuir and LB monolayers, both of which correspond to a similar surface pressure, the LB monolayer tended to have fewer gauche defects and have less tilted terminal methyls in the n-pentadecyl groups than the corresponding Langmuir monolayer. In addition, weak vibrational bands, which have been hardly seen by the conventional (homodyne-detected) VSFG spectroscopy, were clearly observed with their phases, or arguments, for the first time.

Mechanism of vibrational energy dissipation of free OH groups at the air-water interface.

Interfaces of liquid water play a critical role in a wide variety of processes that occur in biology, a variety of technologies, and the environment. Many macroscopic observations clarify that the properties of liquid water interfaces significantly differ from those of the bulk liquid. In addition to interfacial molecular structure, knowledge of the rates and mechanisms of the relaxation of excess vibrational energy is indispensable to fully understand physical and chemical processes of water and aqueous solutions, such as chemical reaction rates and pathways, proton transfer, and hydrogen bond dynamics. Here we elucidate the rate and mechanism of vibrational energy dissipation of water molecules at the air-water interface using femtosecond two-color IR-pump/vibrational sum-frequency probe spectroscopy. Vibrational relaxation of nonhydrogen-bonded OH groups occurs at a subpicosecond timescale in a manner fundamentally different from hydrogen-bonded OH groups in bulk, through two competing mechanisms: intramolecular energy transfer and ultrafast reorientational motion that leads to free OH groups becoming hydrogen bonded. Both pathways effectively lead to the transfer of the excited vibrational modes from free to hydrogen-bonded OH groups, from which relaxation readily occurs. Of the overall relaxation rate of interfacial free OH groups at the air-H2O interface, two-thirds are accounted for by intramolecular energy transfer, whereas the remaining one-third is dominated by the reorientational motion. These findings not only shed light on vibrational energy dynamics of interfacial water, but also contribute to our understanding of the impact of structural and vibrational dynamics on the vibrational sum-frequency line shapes of aqueous interfaces.

Sensitive and quantitative probe of molecular chirality with heterodyne-detected doubly resonant sum frequency generation spectroscopy.

Heterodyne-detected vibrationally electronically doubly resonant chiral sum frequency generation (HD-DR chiral SFG) spectroscopy has been developed for the study of chiral molecules with chromophores. The method enables us to detect and distinguish chiral molecules with high sensitivity and to obtain information on molecular vibrations. Strong enhancement due to the electronic resonance improves the sensitivity, and heterodyne detection ensures that the signal intensity is linear to the sample concentration. Detection of HD-DR chiral SFG signal from a dilute solution of binaphthol with 20 mM concentration and tens of nanometers thickness was demonstrated. Taking advantage of the enantiomer-dependent sign and linearity of the signal to the concentration, molecular concentrations and enantiomeric excesses were accurately evaluated. HD-DR chiral SFG is expected to have widespread application in the study of molecular chirality of thin films or samples of a very small quantity.

Interaction of a patterned amphiphilic polyphenylene dendrimer with a lipid monolayer: electrostatic interactions dominate.

Dendrimeric macromolecules with defined shape and size are promising candidates for delivering drug or DNA molecules into cells. In this work we study the influence of an amphiphilic polyphenylene dendrimer on a model cell membrane consisting of a condensed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. A small surface pressure decrease is observed when the dendrimer solution is injected into the aqueous phase below the monolayer. X-ray reflectivity measurements show that the surface monolayer remains intact. The molecular-scale picture is obtained with sum-frequency generation spectroscopy. With this technique, we observe that the tails of the surfactant molecules become less ordered upon interaction with the amphiphilic polyphenylene dendrimer. In contrast, the water molecules below the DPPC layer become more ordered. Our observations suggest that electrostatic interactions between the negative charge of the dendrimer and the positively charged part of the DPPC headgroup keep the dendrimer located below the headgroup. No evidence of dendrimer insertion into the membrane has been observed. Apparently before entering the cell membrane the dendrimer can stick at the hydrophilic part of the lipids.

Communication: Salt-induced water orientation at a surface of non-ionic surfactant in relation to a mechanism of Hofmeister effect.

The behavior of water molecules at the surface of nonionic surfactant (monomyristolein) and effects of monovalent ions on the behavior are investigated using the heterodyne-detected vibrational sum frequency generation spectroscopy. It is found that water molecules at the surface are oriented with their hydrogen atoms pointing to the bulk, and that the degree of orientation depends on the anion strongly but weakly on the cation. With measured surface potentials in those saline solutions, it is concluded that the heterogeneous distribution of anions and cations in combination with the nonionic surfactant causes the water orientation. This heterogeneous distribution well explains the contrasting order of anions and cations with respect to the ion size in the Hofmeister series.

Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes.

The regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin, hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellular origin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However, when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins and immunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantly lowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 from the homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FA release, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on the surface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.

Aqueous heterogeneity at the air/water interface revealed by 2D-HD-SFG spectroscopy.

Water molecules interact strongly with each other through hydrogen bonds. This efficient intermolecular coupling causes strong delocalization of molecular vibrations in bulk water. We study intermolecular coupling at the air/water interface and find intermolecular coupling 1) to be significantly reduced and 2) to vary strongly for different water molecules at the interface--whereas in bulk water the coupling is homogeneous. For strongly hydrogen-bonded OH groups, coupling is roughly half of that of bulk water, due to the lower density in the near-surface region. For weakly hydrogen-bonded OH groups that absorb around 3500 cm(-1), which are assigned to the outermost, yet hydrogen-bonded OH groups pointing towards the liquid, coupling is further reduced by an additional factor of 2. Remarkably, despite the reduced structural constraints imposed by the interfacial hydrogen-bond environment, the structural relaxation is slow and the intermolecular coupling of these water molecules is weak.

Characterization of Secondary Structures of Model Polypeptides in Solutions with Hyper-Raman Spectroscopy.

Characterization of the secondary structures of two model polypeptides, poly-l-lysine and poly-l-glutamic acid in aqueous solutions has been demonstrated by hyper-Raman (HR) spectroscopy for the first time. Complementary to infrared (IR) and visible Raman spectroscopy, HR spectroscopy gives the amide I, II, and III bands originating from the polypeptide backbones and the CCH3 symmetric bending mode, enabling us to distinguish different conformations. The α-helix gives the broad and weak amide III band, while the ß-sheet and the random coil show similar spectral patterns with different relative intensities between the amide I and II bands. HR spectra from aqueous solutions of the α-helix and the random coil of poly-l-ornithine also possess these spectral features. The HR spectra are analogous to UV resonance Raman (UVRR) spectra, indicating the signal enhancement due to the electronic resonance effect via the π-π* transition. In contrast, the vibrational frequencies of the amide I band in the HR spectra are much higher than those in the IR, visible Raman, and UVRR spectra, suggesting the non-coincidence between HR, IR, and Raman bands. Our finding suggests that HR spectroscopy is promising to provide complementary information on the secondary structures of polypeptides in aqueous solutions as a spectral approach differing from existing vibrational spectroscopic methods.

Is Unified Understanding of Vibrational Coupling of Water Possible? Hyper-Raman Measurement and Machine Learning Spectra.

The impact of the vibrational coupling of the OH stretch mode on the spectra differs significantly between IR and Raman spectra of water. Unified understanding of the vibrational couplings is not yet achieved. By using a different class of vibrational spectroscopy, hyper-Raman (HR) spectroscopy, together with machine-learning-assisted HR spectra calculation, we examine the impact of the vibrational couplings of water through the comparison of isotopically diluted H2O and pure H2O. We found that the isotopic dilution reduces the HR bandwidths, but the impact of the vibrational coupling is smaller than in the IR and parallel-polarized Raman. Machine learning HR spectra indicate that the intermolecular coupling plays a major role in broadening the bandwidth, while the intramolecular coupling is negligibly small, which is consistent with the IR and Raman spectra. Our result clearly demonstrates a limited impact of the intramolecular vibration, independent of the selection rules of vibrational spectroscopies.

Label-free tetra-modal molecular imaging of living cells with CARS, SHG, THG and TSFG (coherent anti-Stokes Raman scattering, second harmonic generation, third harmonic generation and third-order sum frequency generation).

We have developed a new multimodal molecular imaging system that combines CARS (coherent anti-Stokes Raman scattering), SHG (second harmonic generation), THG (third harmonic generation) and multiplex TSFG (third-order sum frequency generation) using a subnanosecond white-light laser source. Molecular composition and their distribution in living cells are clearly visualized with different contrast enhancements through different mechanisms of CARS, SHG, THG and TSFG. A correlation image of CARS and TSF reveals that the TSF signal is generated predominantly from lipid droplets inside a cell as well as the peripheral cell wall.

Time-frequency resolved analysis of a nanosecond supercontinuum source dedicated to multiplex CARS application.

In this paper, we describe and investigate the properties of a broadband source designed from a nanosecond microchip laser operating at high repetition rate and dedicated to multiplex-CARS application. We demonstrate that a strong reshaping of the initial pulse profile drastically affects the Stokes wave and therefore represents an important limitation in CARS experiment. In particular, we emphasize the saturation effect of the peak power of the Stokes wave resulting from supercontinuum generation. However, we show that this type of compact system can be particularly suitable for achieving CARS measurement.

Observation of Raman optical activity by heterodyne-detected polarization-resolved coherent anti-Stokes Raman scattering.

We report the first observation of Raman optical activity (ROA) by coherent anti-Stokes Raman scattering. Thanks to the more freedom of polarization configurations in coherent anti-Stokes Raman scattering than in spontaneous Raman spectroscopy, the contrast ratio of the chiral signal to the achiral background has been improved markedly. For (-)-ß-pinene, it is 2 orders of magnitude better than that in the reported spontaneous ROA measurement. This is also the first measurement of ROA signal using a pulsed laser source.

Hyper-Raman Spectroscopic Investigation of Amide Bands of N-Methylacetamide in Liquid/Solution Phase.

We have demonstrated hyper-Raman (HR) spectroscopy of N-methylacetamide (NMA) for the first time. Fundamental knowledge of amide bands in HR spectra has been obtained. HR spectra of NMA exhibit various amide bands with different intensity patterns from Raman and IR spectra. The amide III and II signals were strongly observed, suggesting the possible application of HR spectroscopy to analyze secondary structures, complementary to IR and Raman spectroscopy. The peak positions of HR amide bands sharply reflect the hydrogen-bonding environment around the molecule. The depolarization ratios of the amide II and III bands at 532 nm excitation suggest the resonance HR effect via the π-π* transition. In contrast, that of the amide I band of neat NMA indicates the contribution of high energy transitions to its signal enhancement. This work proposes that HR spectroscopy can be a powerful tool for studying the molecular structure and environment of biomolecules with peptide bonds.

Interfacial Water Structure of Binary Liquid Mixtures Reflects Nonideal Behavior.

The evaporation of molecules from water-organic solute binary mixtures is key for both atmospheric and industrial processes such as aerosol formation and distillation. Deviations from ideal evaporation energetics can be assigned to intermolecular interactions in solution, yet evaporation occurs from the interface, and the poorly understood interfacial, rather than the bulk, structure of binary mixtures affects evaporation kinetics. Here we determine the interfacial structure of nonideal binary mixtures of water with methanol, ethanol, and formic acid, by combining surface-specific vibrational spectroscopy with molecular dynamics simulations. We find that the free, dangling OH groups at the interfaces of these differently behaving nonideal mixtures are essentially indistinguishable. In contrast, the ordering of hydrogen-bonded interfacial water molecules differs substantially at these three interfaces. Specifically, the interfacial water molecules become more disordered (ordered) in mixtures with methanol and ethanol (formic acid), showing higher (lower) vapor pressure than that predicted by Raoult's law.