Spiral packing and chiral selectivity in model membranes probed by phase-resolved sum-frequency generation microscopy.

Since the lipid raft model was developed at the end of the last century, it became clear that the specific molecular arrangements of phospholipid assemblies within a membrane have profound implications in a vast range of physiological functions. Studies of such condensed lipid islands in model systems using fluorescence and Brewster angle microscopies have shown a wide range of sizes and morphologies, with suggestions of substantial in-plane molecular anisotropy and mesoscopic structural chirality. Whilst these variations can significantly alter many membrane properties including its fluidity, permeability and molecular recognition, the details of the in-plane molecular orientations underlying these traits remain largely unknown. Here, we use phase-resolved sum-frequency generation microscopy on model membranes of mixed chirality phospholipid monolayers to fully determine the three-dimensional molecular structure of the constituent micron-scale condensed domains. We find that the domains possess curved molecular directionality with spiralling mesoscopic packing, where both the molecular and spiral turning directions depend on the lipid chirality, but form structures clearly deviating from mirror symmetry for different enantiomeric mixtures. This demonstrates strong enantioselectivity in the domain growth process and indicates fundamental thermodynamic differences between homo- and heterochiral membranes, which may be relevant in the evolution of homochirality in all living organisms.

Obtaining extended insight into molecular systems by probing multiple pathways in second-order nonlinear spectroscopy.

Second-order nonlinear spectroscopy is becoming an increasingly important technique in the study of interfacial systems owing to its marked ability to study molecular structures and interactions. The properties of such a system under investigation are contained within their intrinsic second-order susceptibilities which are mapped onto the measured nonlinear signals (e.g. sum-frequency generation) through the applied experimental settings. Despite this yielding a plethora of information, many crucial aspects of molecular systems typically remain elusive, for example the depth distributions, molecular orientation and local dielectric properties of its constituent chromophores. Here, it is shown that this information is contained within the phase of the measured signal and, critically, can be extracted through measurement of multiple nonlinear pathways (both the sum-frequency and difference-frequency output signals). Furthermore, it is shown that this novel information can directly be correlated to the characteristic vibrational spectra, enabling a new type of advanced sample characterization and a profound analysis of interfacial molecular structures. The theory underlying the different contributions to the measured phase of distinct nonlinear pathways is derived, after which the presented phase disentanglement methodology is experimentally demonstrated for model systems of self-assembled monolayers on several metallic substrates. The obtained phases of the local fields are compared to the corresponding phases of the nonlinear Fresnel factors calculated through the commonly used theoretical model, the three-layer model. It is found that, despite its rather crude assumptions, the model yields remarkable similarity to the experimentally obtained values, thus providing validation of the model for many sample classes.

Compact oblique-incidence nonlinear widefield microscopy with paired-pixel balanced imaging.

Nonlinear (vibrational) microscopy has emerged as a successful tool for the investigation of molecular systems as it combines label-free chemical characterization with spatial resolution on the sub-micron scale. In addition to the molecular recognition, the physics of the nonlinear interactions allows in principle to obtain structural information on the molecular level such as molecular orientations. Due to technical limitations such as the relatively complex imaging geometry with the required oblique sample irradiation and insufficient sensitivity of the instrument this detailed molecular information is typically not accessible using widefield imaging. Here, we present, what we believe to be, a new microscope design that addresses both challenges. We introduce a simplified imaging geometry that enables the measurement of distortion-free widefield images with free space oblique sample irradiation achieving high spatial resolution (∼1 µm). Furthermore, we present a method based on a paired-pixel balanced detection system for sensitivity improvement. With this technique, we demonstrate a substantial enhancement of the signal-to-noise ratio of up to a factor of 10. While both experimental concepts presented in this work are very general and can, in principle, be applied to various microscopy techniques, we demonstrate their performance for the specific case of heterodyned, sum frequency generation (SFG) microscopy.

A General Approach To Combine the Advantages of Collinear and Noncollinear Spectrometer Designs in Phase-Resolved Second-Order Nonlinear Spectroscopy.

Recent years have seen a huge progress in the development of phase-sensitive second-order laser spectroscopy which has proven to be a very powerful tool for the investigation of interfaces. In these techniques, the nonlinear interaction between two short laser pulses and the sample yields a signal pulse which subsequently interferes with a third pulse, the so-called local oscillator. To obtain accurate phase information, the relative phases between the signal and local oscillator pulses must be stabilized and their timings precisely controlled. Despite much progress made, fulfilling both requirements remains a formidable experimental challenge. The two common approaches employ different beam geometries which each yields its particular advantages and deficiencies. While noncollinear spectrometers allow for a relatively simple timing control they typically yield poor phase stability and require a challenging alignment. Collinear approaches in contrast come with a simplified alignment and improved phase stability but typically suffer from a highly limited timing control. In this contribution we present a general experimental solution which allows for combining the advantages of both approaches while being compatible with most of the common spectrometer types. On the basis of a collinear geometry, we exploit different selected polarization states of the light pulses in well-defined places in the spectrometer to achieve a precise timing control. The combination of this technique with a balanced detection scheme allows for the acquisition of highly accurate phase-resolved nonlinear spectra without any loss in experimental flexibility. In fact, we show that the implementation of this technique allows us to employ advanced pulse timing schemes inside the spectrometer, which can be used to suppress nonlinear background signals and extend the capabilities of our spectrometer to measure phase-resolved sum frequency spectra of interfaces in a liquid cell.

Detecting weak signals from interfaces by high accuracy phase-resolved SFG spectroscopy.

Much work over the last 25 years has demonstrated that the interface-specific, all-optical technique, vibrational sum frequency generation (v-SFG) spectroscopy, is often uniquely capable of characterizing the structure and dynamics of interfacial species. The desired information in such a measurement is the complex second order susceptibility which gives rise to the nonlinear response from interfacial molecules. The ability to detect molecular species yielding only small contributions to the susceptibility is meanwhile limited by the precision by which the spectral phase and amplitude can be determined. In this study we describe a new spectrometer design that offers unprecedented phase and amplitude accuracy for extended studies that involve multiple spectral acquisitions while modifying sample properties. The key to this significant improvement to the sensitivity of the technique is the combination of a full collinear beam geometry with broadband spectral sampling and the ability to simultaneously measure the complex sample and reference spectrum. We show that using this technique uncertainties in the reference phase and amplitude can be greatly reduced. Furthermore, we show that using balanced detection, the signal to noise ratio can be increased by one order of magnitude. The capabilities of the spectrometer are demonstrated by the isolation of a small isotropic surface signal from the bulk dominated nonlinear optical response of z-cut quartz. The achieved precision of our spectrometer enables measurements not currently feasible in v-SFG spectroscopy.

Hydrophobic Water Probed Experimentally at the Gold Electrode/Aqueous Interface.

Quantitative description of reaction mechanisms in aqueous phase electrochemistry requires experimental characterization of local water structure at the electrode/aqueous interface and its evolution with changing potential. Gaining such insight experimentally under electrochemical conditions is a formidable task. The potential-dependent structure of a subpopulation of interfacial water with one OH group pointing towards a gold working electrode is characterized using interface specific vibrational spectroscopy in a thin film electrochemical cell. Such free-OH groups are the molecular level observable of an extended hydrophobic interface. This free-OH interacts only weakly with the Au surface at all potentials, has an orientational distribution that narrows approaching the potential of zero charge, and disappears on oxidation of the gold electrode.

Anharmonic exciton dynamics and energy dissipation in liquid water from two-dimensional infrared spectroscopy.

Water's extended hydrogen-bond network results in rich and complex dynamics on the sub-picosecond time scale. In this paper, we present a comprehensive analysis of the two-dimensional infrared (2D IR) spectrum of O-H stretching vibrations in liquid H2O and their interactions with bending and intermolecular vibrations. By exploring the dependence of the spectrum on waiting time, temperature, and laser polarization, we refine our molecular picture of water's complex ultrafast dynamics. The spectral evolution following excitation of the O-H stretching resonance reveals vibrational dynamics on the 50-300 fs time scale that are dominated by intermolecular delocalization. These O-H stretch excitons are a result of the anharmonicity of the nuclear potential energy surface that arises from the hydrogen-bonding interaction. The extent of O-H stretching excitons is characterized through 2D depolarization measurements that show spectrally dependent delocalization in agreement with theoretical predictions. Furthermore, we show that these dynamics are insensitive to temperature, indicating that the exciton dynamics alone set the important time scales in the system. Finally, we study the evolution of the O-H stretching mode, which shows highly non-adiabatic dynamics suggestive of vibrational conical intersections. We argue that the so-called heating, commonly observed within ∼1 ps in nonlinear IR spectroscopy of water, is a nonequilibrium state better described by a kinetic temperature rather than a Boltzmann distribution. Our conclusions imply that the collective nature of water vibrations should be considered in describing aqueous solvation.

Protons and Hydroxide Ions in Aqueous Systems.

Understanding the structure and dynamics of water's constituent ions, proton and hydroxide, has been a subject of numerous experimental and theoretical studies over the last century. Besides their obvious importance in acid-base chemistry, these ions play an important role in numerous applications ranging from enzyme catalysis to environmental chemistry. Despite a long history of research, many fundamental issues regarding their properties continue to be an active area of research. Here, we provide a review of the experimental and theoretical advances made in the last several decades in understanding the structure, dynamics, and transport of the proton and hydroxide ions in different aqueous environments, ranging from water clusters to the bulk liquid and its interfaces with hydrophobic surfaces. The propensity of these ions to accumulate at hydrophobic surfaces has been a subject of intense debate, and we highlight the open issues and challenges in this area. Biological applications reviewed include proton transport along the hydration layer of various membranes and through channel proteins, problems that are at the core of cellular bioenergetics.

Ultrafast 2D IR spectroscopy of the excess proton in liquid water.

Despite decades of study, the structures adopted to accommodate an excess proton in water and the mechanism by which they interconvert remain elusive. We used ultrafast two-dimensional infrared (2D IR) spectroscopy to investigate protons in aqueous hydrochloric acid solutions. By exciting O-H stretching vibrations and detecting the spectral response throughout the mid-IR region, we observed the interaction between the stretching and bending vibrations characteristic of the flanking waters of the Zundel complex, [H(H2O)2](+), at 3200 and 1760 cm(-1), respectively. From time-dependent shifts of the stretch-bend cross peak, we determined a lower limit on the lifetime of this complex of 480 femtoseconds. These results suggest a key role for the Zundel complex in aqueous proton transfer.

Plasmons in supported size-selected silver nanoclusters.

The plasmonic behavior of size-selected supported silver clusters is studied by surface second harmonic generation spectroscopy for the first time. A blue shift of ∼0.2 eV in the plasmon resonance is observed with decreasing cluster size from Ag55 to Ag9. In addition to the general blue shift, a nonscalable size-dependence is also observed in plasmonic behavior of Ag nanoclusters, which is attributed to varying structural properties of the clusters. The results are in quantitative agreement with a hybrid theoretical model based on Mie theory and the existing DFT calculations.

Direct observation of intermolecular interactions mediated by hydrogen bonding.

Although intermolecular interactions are ubiquitous in physicochemical phenomena, their dynamics have proven difficult to observe directly, and most experiments rely on indirect measurements. Using broadband two-dimensional infrared spectroscopy (2DIR), we have measured the influence of hydrogen bonding on the intermolecular vibrational coupling between dimerized N-methylacetamide molecules. In addition to strong intramolecular coupling between N-H and C=O oscillators, cross-peaks in the broadband 2DIR spectrum appearing upon dimerization reveal strong intermolecular coupling that changes the character of the vibrations. In addition, dimerization changes the effects of intramolecular coupling, resulting in Fermi resonances between high and low-frequency modes. These results illustrate how hydrogen bonding influences the interplay of inter- and intramolecular vibrations, giving rise to correlated nuclear motions and significant changes in the vibrational structure of the amide group. These observations have direct impact on modeling and interpreting the IR spectra of proteins. In addition, they illustrate a general approach to direct molecular characterization of intermolecular interactions.

Orientational changes of supported chiral 2,2'-dihydroxy-1,1'binaphthyl molecules.

Well defined thin molecular films of 2,2'-dihydroxy-1,1'binaphthyl (binol) molecules at coverages between 5 × 10(15) molecules per cm(2) and 10(17) molecules per cm(2) on thin glass (BK7) substrates were investigated under ultra-high-vacuum (UHV) conditions. Second-Harmonic-Generation Optical-Rotatory-Dispersion measurements (SHG-ORD) were performed using a dedicated spectroscopic setup which allows for the determination of the rotation angle of the SH-signal of two enantiomers. Rotation angles of up to 38 degrees were measured. The chirality of the two enantiomers has been studied at 674 nm (337 nm resonance wavelength) in the transmission mode. Coverage dependent orientation evolution of binol molecular films was revealed by precise monitoring of the surface coverage while performing SHG-ORD experiments. We show that the molecules reach their final orientation at a surface coverage of 5 × 10(16) molecules per cm(2). From the obtained experimental data the ratio of chiral and achiral susceptibility components could be calculated and was observed to change with coverage.