pH drives electron density fluctuations that enhance electric field-induced liquid flow.

Liquid flow along a charged interface is commonly described by classical continuum theory, which represents the electric double layer by uniformly distributed point charges. The electrophoretic mobility of hydrophobic nanodroplets in water doubles in magnitude when the pH is varied from neutral to mildly basic (pH 7 → 11). Classical continuum theory predicts that this increase in mobility is due to an increased surface charge. Here, by combining all-optical measurements of surface charge and molecular structure, as well as electronic structure calculations, we show that surface charge and molecular structure at the nanodroplet surface are identical at neutral and mildly basic pH. We propose that the force that propels the droplets originates from two factors: Negative charge on the droplet surface due to charge transfer from and within water, and anisotropic gradients in the fluctuating polarization induced by the electric field. Both charge density fluctuations couple with the external electric field, and lead to droplet flow. Replacing chloride by hydroxide doubles both the charge conductivity via the Grotthuss mechanism, and the droplet mobility. This general mechanism deeply impacts a plethora of processes in biology, chemistry, and nanotechnology and provides an explanation of how pH influences hydrodynamic phenomena and the limitations of classical continuum theory currently used to rationalize these effects.

Interfacial Inversion, Interference, and IR Absorption in Vibrational Sum Frequency Scattering Experiments.

Molecular interfacial structure greatly determines the properties of nano- and microscale systems. Vibrational sum frequency scattering (SFS) spectroscopy is a unique interface-selective tool to measure the interfacial vibrational spectrum of sub-micron to micron-scale objects dispersed in liquid and solid media. The interfacial structure is extracted from the interfacial susceptibility, a physical property derived from the intensity. Here, we describe the effect of infrared absorption that occurs in a bulk medium that is spectroscopically complex and use the results to investigate the effects of interfacial inversion, interfacial interference, and interfacial interference combined with absorption. We use the same three chemicals to do so, hexadecane oil, water, and a neutral Span80 surfactant. For all cases, the effective surface susceptibility can be retrieved from the intensity. We further find that inverting the phases results in different interfacial structures, even though they are composed of the same three chemicals, and explain this in terms of the different interactions that are necessary to stabilize the drops: steric stabilization for water drops in oil vs. charge stabilization for oil drops in water. Interfacial interference can be used to estimate the surface density of different compounds.

High throughput wide field second harmonic imaging of giant unilamellar vesicles.

Cell-sized giant unilamellar vesicles (GUVs) are an ideal tool for understanding lipid membrane structure and properties. Label-free spatiotemporal images of their membrane potential and structure would greatly aid the quantitative understanding of membrane properties. In principle, second harmonic imaging is a great tool to do so, but the low degree of spatial anisotropy that arises from a single membrane limits its application. Here, we advance the use of wide-field high throughput SH imaging by SH imaging with the use of ultrashort laser pulses. We achieve a throughput improvement of 78% of the maximum theoretical value and demonstrate subsecond image acquisition times. We show how the interfacial water intensity can be converted into a quantitative membrane potential map. Finally, for GUV imaging, we compare this type of nonresonant SH imaging to resonant SH imaging and two photon imaging using fluorophores.

Water Structure at the Hydrophobic Nanodroplet Surface Revealed by Vibrational Sum Frequency Scattering Using Isotopic Dilution.

The water structure at the hydrophobic/water interface is key toward understanding hydrophobicity at the molecular level. Herein, we characterize the hydrogen-bonding network of interfacial water next to sub-micron-sized hydrophobic oil droplets dispersed in water using isotopic dilution vibrational sum frequency scattering (SFS) spectroscopy. The relative intensity of different modes, the frequency shift of the uncoupled O-D spectrum, and a low-frequency shoulder (2395 cm-1) reveal that water forms an overall stronger hydrogen-bonding network next to hydrophobic droplets compared to bulk water and the air/water interface. Half of the spectral width of the oil droplet SFS spectrum is determined by inter- and intramolecular coupling of water molecules. Isotopic dilution also confirms the presence of a broad distribution (ca. 2640-2745 cm-1) of non-water-hydrogen-bonded O-D modes that are red-shifted and broadened compared to similar species at the air/water interface. This band corroborates the presence of charge transfer between water and oil.

Ultrasensitive Label-Free Detection of Protein-Membrane Interaction Exemplified by Toxin-Liposome Insertion.

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with Kd = (1.5 ± 0.2) × 10-12 M. Our results demonstrate a promising approach to probe protein-membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (<10 µL). These findings enable the detection of low-abundance proteins and their interaction with membranes.

Hyaluronan orders water molecules in its nanoscale extended hydration shells.

Hyaluronan (HA) is an anionic, highly hydrated bio-polyelectrolyte found in the extracellular environment, like the synovial fluid between joints. We explore the extended hydration shell structure of HA in water using femtosecond elastic second-harmonic scattering (fs-ESHS). HA enhances orientational water-water correlations. Angle-resolved fs-ESHS measurements and nonlinear optical modeling show that HA behaves like a flexible chain surrounded by extended shells of orientationally correlated water. We describe several ways to determine the concentration-dependent size and shape of a polyelectrolyte in water, using the amount of water oriented by the polyelectrolyte charges as a contrast agent. The spatial extent of the hydration shell is determined via temperature-dependent measurements and can reach up to 475 nm, corresponding to a length of 1600 water molecules. A strong isotope effect, stemming from nuclear quantum effects, is observed when light water (H2O) is replaced by heavy water (D2O), amounting to a factor of 4.3 in the scattered SH intensity.

Self-Assembly at Water Nanodroplet Interfaces Quantified with Nonlinear Light Scattering.

The interfaces of water micro- and nanodroplets drive environmental, medical, catalytic, biological, and chemical biphasic processes. The interfacial droplet structure and electrostatics greatly determine the reactivity and efficiency of these processes. Droplet interfacial properties are elusive and generally inferred from bulk measurements and are therefore anything but exact. Here, we quantify the interfacial ordering of water and the electrostatic surface potential of nanoscale water droplets in an apolar liquid using angle-resolved polarimetric second-harmonic scattering. We also present a method to determine the amount of free charges in the hydrophobic phase, reaching a sensitivity that is 3 orders of magnitude better than conductivity measurements. Investigating the structural and surface electrostatic changes induced by AOT surfactant adsorption, we find that both the hydrogen bonding as well as the electrostatics strongly depend on the surfactant concentration. Above the critical micelle concentration, the interface mediates micelle self-assembly.

On the stability and necessary electrophoretic mobility of bare oil nanodroplets in water.

Hydrophobic oil droplets, particles, and air bubbles can be dispersed in water as kinetically stabilized dispersions. It has been established since the 19th century that such objects harbor a negative electrostatic potential roughly twice larger than the thermal energy. The source of this charge continues to be one of the core observations in relation to hydrophobicity, and its molecular explanation is still debated. What is clear though is that the stabilizing interaction in these systems is understood in terms of electrostatic repulsion via Derjaguin, Landau, Verwey, and Overbeek theory. Recent work [A. P. Carpenter et al., Proc. Natl. Acad. Sci. U. S. A. 116, 9214 (2019)] has added another element into the discussion, reporting the creation of bare near-zero charged droplets of oil in neat water that are stable for several days. Key to the creation of the droplets is a rigorous glassware cleaning procedure. Here, we investigate these conclusions and show that the cleaning procedure of glassware has no influence on the electrophoretic mobility of the droplets and that oil droplets with near-zero charge are unstable. We provide an alternative possible explanation for the observations involving glass surface chemistry.

Transient domains of ordered water induced by divalent ions lead to lipid membrane curvature fluctuations.

Cell membranes are composed of a hydrated lipid bilayer that is molecularly complex and diverse, and the link between molecular hydration structure and membrane macroscopic properties is not well understood, due to a lack of technology that can probe and relate molecular level hydration information to micro- and macroscopic properties. Here, we demonstrate a direct link between lipid hydration structure and macroscopic dynamic curvature fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we observe the formation of transient domains of ordered water at the interface of freestanding lipid membranes. These domains are induced by the binding of divalent ions and their structure is ion specific. Using nonlinear optical theory, we convert the spatiotemporal SH intensity into maps of membrane potential, surface charge density, and binding free energy. Using an electromechanical theory of membrane bending, we show that transient electric field gradients across the membrane induce spatiotemporal membrane curvature fluctuations.

Polyelectrolytes induce water-water correlations that result in dramatic viscosity changes and nuclear quantum effects.

Ions interact with water via short-ranged ion-dipole interactions. Recently, an additional unexpected long-ranged interaction was found: The total electric field of ions influences water-water correlations over tens of hydration shells, leading to the Jones Ray effect, a 0.3% surface tension depression. Here, we report such long-range interactions contributing substantially to both molecular and macroscopic properties. Femtosecond elastic second harmonic scattering (fs-ESHS) shows that long-range electrostatic interactions are remarkably strong in aqueous polyelectrolyte solutions, leading to an increase in water-water correlations. This increase plays a role in the reduced viscosity, which changes more than two orders of magnitude with polyelectrolyte concentration. Using D2O instead of H2O shifts both the fs-ESHS and the viscosity curve by a factor of ~10 and reduces the maximum viscosity value by 20 to 300%, depending on the polyelectrolyte. These phenomena cannot be explained using a mean-field approximation of the solvent and point to nuclear quantum effects.

Membrane water for probing neuronal membrane potentials and ionic fluxes at the single cell level.

Neurons communicate through electrochemical signaling within a complex network. These signals are composed of changes in membrane potentials and are traditionally measured with the aid of (toxic) fluorescent labels or invasive electrical probes. Here, we demonstrate an improvement in label-free second harmonic neuroimaging sensitivity by ~3 orders of magnitude using a wide-field medium repetition rate illumination. We perform a side-by-side patch-clamp and second harmonic imaging comparison to demonstrate the theoretically predicted linear correlation between whole neuron membrane potential changes and the square root of the second harmonic intensity. We assign the ion induced changes to the second harmonic intensity to changes in the orientation of membrane interfacial water, which is used to image spatiotemporal changes in the membrane potential and K+ ion flux. We observe a non-uniform spatial distribution and temporal activity of ion channels in mouse brain neurons.

Comment on "Water-water correlations in electrolyte solutions probed by hyper-Rayleigh scattering" [J. Chem. Phys. 147, 214505 (2017)].

The work by Shelton [J. Chem. Phys. 147, 214505 (2017)] discussed and interpreted differences with a previous study by Chen et al. [Sci. Adv. 2, e1501891 (2016)] regarding the influence of electrolytes on the structure of water. It is argued by Shelton [J. Chem. Phys. 147, 214505 (2017)] that impurities and hyper-Raman scattering contributions are the reasons for differences in the measured second harmonic intensity between the above two studies. Here, we show that these proposed effects are not relevant and discuss the influence of pulse parameters, focusing on pulse duration, since these two sets of experiments are performed with substantially different pulse durations, 100 ns and 190 fs, respectively. We show that inelastic higher-order effects play a role in the experiment with 100 ns laser pulses (the probed structure is that of the electrolyte solution that is modified by a laser pulse), while in the experiment with 190 fs laser pulses, only the elastic second-order response is measured (probing the unperturbed water structure).

Freezing effects of oil-in-water emulsions studied by sum-frequency scattering spectroscopy.

Temperature-dependent sum-frequency scattering spectroscopy is used to study the properties of hexadecane and dodecane oil droplets in water. The sum-frequency scattering spectra contain vibrational bands that correspond to the symmetric and antisymmetric CH stretching vibrations of the methylene (CH2) and methyl (CH3) groups of the alkane molecules. The relative amplitudes of the vibrational bands provide information on the surface structure and the shape of the oil droplets. We study the sum-frequency scattering spectra over a temperature range of -48 to 24 °C, including the freezing transitions of the water matrix and the oil droplets. Hexadecane oil droplets freeze at a higher temperature than the surrounding water, whereas dodecane oil droplets freeze at a lower temperature than the surrounding water. This allows us to independently study the freezing effect of oil and water on the surface structure of the oil droplets. In both cases, freezing leads to a change in the polarization dependencies that are valid in the case of the spherical-symmetric shapes that the oil droplets assume when both water and oil are liquid. We find that the freezing of water leads to a strong distortion of the liquid dodecane surface but has little effect on the surface of already solidified hexadecane. For completely frozen emulsions a further decrease in temperature is observed to lead to a further distortion of the surface of the solid oil particles, which might be caused by increasing hardness of the ice matrix encapsulating the particles.

Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells.

Demands of higher spatial and temporal resolutions in linear and nonlinear imaging keep pushing the limits of optical microscopy. We showed recently that a multiphoton microscope with 200 kHz repetition rate and wide-field illumination has a 2-3 orders of magnitude improved throughput compared to a high repetition rate confocal scanning microscope. Here, we examine the photodamage mechanisms and thresholds in live cell imaging for both systems. We first analyze theoretically the temperature increase in an aqueous solution resulting from illuminating with different repetition rates (keeping the deposited energy and irradiated volume constant). The analysis is complemented with photobleaching experiments of a phenolsulfonphthalein (phenol red) solution. Combining medium repetition rates and wide-field illumination promotes thermal diffusivity, which leads to lower photodamage and allows for higher peak intensities. A three day proliferation assay is also performed on living cells to confirm these results: dwell times can be increased by a factor of 3×10(6) while still preserving cell proliferation. By comparing the proliferation data with the endogenous two-photon fluorescence decay, we propose to use the percentage of the remaining endogenous two-photon fluorescence after exposure as a simple in-situ viability test. These findings enable the possibility of long-term imaging and reduced photodamage.

Detection of buried microstructures by nonlinear light scattering spectroscopy.

Many processes in chemistry and physics rely on the structure, growth or change of material buried in solids. The impenetrable surrounding medium often prohibits the study of such material in situ. Nonlinear light scattering can be used to observe the internal structure of a crystalline state embedded inside another solid state. Vibrational sum frequency scattering patterns of polymer microspheres, consisting of both amorphous and crystalline material, reveal the size of the buried microstructure and the optical components of the second-order susceptibility of the material. The vibrational spectra reveal the molecular structure.