Dissecting the Membrane Association Mechanism of Aerolysin Pores at Femtomolar Concentrations Using Water as a Probe.

Aerolysin is a bacterial toxin that forms transmembrane pores at the host plasma membrane and has a narrow internal diameter and great stability. These assets make it a highly promising nanopore for detecting biopolymers such as nucleic acids and peptides. Although much is known about aerolysin from a microbiological and structural perspective, its membrane association and pore-formation mechanism are not yet fully understood.

Dissecting the hydrogen bond network of water: Charge transfer and nuclear quantum effects.

The molecular structure of water is dynamic, with intermolecular hydrogen (H) bond interactions being modified by both electronic charge transfer and nuclear quantum effects (NQEs). Electronic charge transfer and NQEs potentially change under acidic or basic conditions, but such details have not been measured. In this work, we developed correlated vibrational spectroscopy, a symmetry-based method that separates interacting from noninteracting molecules in self- and cross-correlation spectra, giving access to previously inaccessible information.

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.

Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations.

Does the ionic distribution in the electrical double layer modify second harmonic scattering?

Surface-specific nonlinear optical techniques are ideally suited to investigate the complex structure of aqueous interfaces. For colloidal particles dispersed in aqueous solutions, interfacial properties can be retrieved with angle-resolved second harmonic scattering (AR-SHS). The mathematical framework of AR-SHS does not require a priori knowledge on the electrostatic distribution in the first few nanometers close to the interface, therefore allowing us to formulate a molecular-level description of the electrical double layer (EDL) based on the experimental data.

Liposomes and Lipid Droplets Display a Reversal of Charge-Induced Hydration Asymmetry.

The unique properties of water are critical for life. Water molecules have been reported to hydrate cations and anions asymmetrically in bulk water, being a key element in the balance of biochemical interactions. We show here that this behavior extends to charged lipid nanoscale interfaces.

Liquid-activated quantum emission from pristine hexagonal boron nitride for nanofluidic sensing.

Liquids confined down to the atomic scale can show radically new properties. However, only indirect and ensemble measurements operate in such extreme confinement, calling for novel optical approaches that enable direct imaging at the molecular level. Here we harness fluorescence originating from single-photon emitters at the surface of hexagonal boron nitride for molecular imaging and sensing in nanometrically confined liquids.

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.

Evolution of the electrical double layer with electrolyte concentration probed by second harmonic scattering.

Investigating the electrical double layer (EDL) structure has been a long-standing challenge and has seen the emergence of several sophisticated techniques able to probe selectively the few molecular layers of a solid/water interface. While a qualitative estimation of the thickness of the EDL can be obtained using simple theoretical models, following experimentally its evolution is not straightforward and can be even more complicated in nano- or microscale systems, particularly when changing the ionic concentration by several orders of magnitude. Here, we bring insight into the structure of the EDL of SiO nanoparticle suspensions and its evolution with increasing ionic concentration using angle-resolved second harmonic scattering (AR-SHS).

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.

Lysozyme Interaction with Phospholipid Nanodroplets Probed by Sum Frequency Scattering Vibrational Spectroscopy.

When a nanoparticle (NP) is introduced into a biological environment, its identity and interactions are immediately attributed to the dense layer of proteins that quickly covers the particle. The formation of this layer, dubbed the protein corona, is in general a combination of proteins interacting with the surface of the NP and a contest between other proteins for binding sites either at the surface of the NP or upon the dense layer. Despite the importance for surface engineering and drug development, the molecular mechanisms and structure behind interfacial biomolecule action have largely remained elusive.

Size dependence of second-harmonic scattering from nanoparticles: Disentangling surface and electrostatic contributions.

Polarimetric angle-resolved second-harmonic scattering (AR-SHS) is an all-optical tool enabling the study of unlabeled interfaces of nano-sized particles in an aqueous solution. As the second harmonic signal is modulated by interference between nonlinear contributions originating at the particle's surface and those originating in the bulk electrolyte solution due to the presence of a surface electrostatic field, the AR-SHS patterns give insight into the structure of the electrical double layer. The mathematical framework of AR-SHS has been previously established, in particular regarding changes in probing depth with ionic strength.

Passive transport of Ca ions through lipid bilayers imaged by widefield second harmonic microscopy.

In biology, release of Ca ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca. For proper understanding of membrane permeability to Ca, both membrane hydration and the structure of the hydrophobic core must be taken into account.

Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes.

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems.

Three-Dimensional Confinement of Water: HO Exhibits Long-Range (>50 nm) Structure while DO Does Not.

Water is the liquid of life thanks to its three-dimensional adaptive hydrogen (H)-bond network. Confinement of this network may lead to dramatic structural changes influencing chemical and physical transformations. Although confinement effects occur on a <1 nm length scale, the upper length scale limit is unknown.

Charge Gradients around Dendritic Voids Cause Nanoscale Inhomogeneities in Liquid Water.

Water is the matrix of life and serves as a solvent for numerous physical and chemical processes. The origins of the nature of inhomogeneities that exist in liquid water and the time scales over which they occur remains an open question. Here, we report femtosecond elastic second harmonic scattering (fs-ESHS) of liquid water in comparison to an isotropic liquid (CCl) and show that water is indeed a nonuniform liquid.

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) reveal that water forms an overall stronger hydrogen-bonding network next to hydrophobic droplets compared to bulk water and the air/water interface.

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.

Charge transfer across C-H⋅⋅⋅O hydrogen bonds stabilizes oil droplets in water.

The hydrophobic–water interface plays a key role in biological interactions. However, both the hydrophobic–water interfacial molecular structure and the origin of the negative zeta potential of hydrophobic droplets in water are heavily contested. We report polarimetric vibrational sum-frequency scattering of the O–D and C-H stretch modes of 200-nanometer hexadecane oil droplets dispersed in water.

Imaging Cu binding to charged phospholipid membranes by high-throughput second harmonic wide-field microscopy.

The interaction of divalent copper ions (Cu) with cell membranes is crucial for a variety of physiological processes of cells, such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies.

Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water.

Morphological and gel-to-liquid phase transitions of lipid membranes are generally considered to primarily depend on the structural motifs in the hydrophobic core of the bilayer. Structural changes in the aqueous headgroup phase are typically not considered, primarily because they are difficult to quantify. Here, we investigate structural changes of the hydration shells around large unilamellar vesicles (LUVs) in aqueous solution, using differential scanning calorimetry (DSC), and temperature-dependent ζ-potential and high-throughput angle-resolved second harmonic scattering measurements (AR-SHS).

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.

Biodegradable Harmonophores for Targeted High-Resolution Tumor Imaging.

Optical imaging probes have played a major role in detecting and monitoring a variety of diseases. In particular, nonlinear optical imaging probes, such as second harmonic generating (SHG) nanoprobes, hold great promise as clinical contrast agents, as they can be imaged with little background signal and unmatched long-term photostability. As their chemical composition often includes transition metals, the use of inorganic SHG nanoprobes can raise long-term health concerns.