Three-Dimensional Confinement of Water: H2O Exhibits Long-Range (>50 nm) Structure while D2O 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. Here, we investigate 3D-confinement over lengths scales ranging from 58-140 nm. By confining water in zwitterionic liposomes of different sizes and measuring the change in H-bond network conformation using second harmonic scattering (SHS), we determined long-range confinement effects in light and heavy water. D2O displays no detectable 3D-confinement effects <58 nm (<3 × 106 D2O molecules). H2O is distinctly different. The vesicle enclosed inner H-bond network has a different conformation compared to the outside network and the SHS response scales with the volume of the confining space. H2O displays confinement effects over distances >100 nm (>2 × 107 H2O molecules).

Spatiotemporal Imaging of Water in Operating Voltage-Gated Ion Channels Reveals the Slow Motion of Interfacial Ions.

Ion channels are responsible for numerous physiological functions ranging from transport to chemical and electrical signaling. Although static ion channel structure has been studied following a structural biology approach, spatiotemporal investigation of the dynamic molecular mechanisms of operational ion channels has not been achieved experimentally. In particular, the role of water remains elusive. Here, we perform label-free spatiotemporal second harmonic (SH) imaging and capacitance measurements of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by aqueous solution on either side. We observe changes in SH intensity upon channel activation that are traced back to changes in the orientational distribution of water molecules that reorient along the field lines of transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. The time scale of these interfacial changes is influenced by the density of ion channels and is subject to a crowding mechanism. Ion transport along cell membranes is often associated with the propagation of electrical signals in neurons. As our study shows that this process is taking place over seconds, a more complex mechanism is likely responsible for the propagation of neuronal electrical signals than just the millisecond movement of ions.

Chemistry of Lipid Membranes from Models to Living Systems: A Perspective of Hydration, Surface Potential, Curvature, Confinement and Heterogeneity.

Lipid membranes provide diverse and essential functions in our cells relating to transport, energy harvesting and signaling. This variety of functions is controlled by the molecular architecture, such as the presence of hydrating water, specific chemical compounds and microscopic structures, such as the local membrane curvature, as well as macroscopic properties, such as the fluidity of the membrane. To understand the chemistry of membranes, ideally one needs access to multiple length scales simultaneously, using probes that are noninvasive, label-free and membrane-interface specific. This dream is generally pursued by following either a top-down approach, introducing labels to real cell membranes or by following a bottom-up approach with well-controlled but simplified membrane monolayer or supported membrane models. This Perspective offers an alternative path that ultimately envisions bringing together both approaches. By using intermediate nano-, micro- and macroscale free-floating membrane systems in combination with novel nonlinear optical methods, one can advance the understanding of realistic membranes on a more fundamental level. Here, we describe recent advances in understanding membrane molecular structure, hydration, electrostatics and the effect of variable length scale, curvature and confinement for 3D nano- and microscale membrane systems such as lipid droplets and liposomes. We also describe an approach to image membrane hydration and membrane potentials in real time and space together with an application to neuroscience. In doing so, we consider the emerging role of interfacial transient structural heterogeneities that are apparent in both model membranes as well as in whole cells.

Interaction of Oil and Lipids in Freestanding Lipid Bilayer Membranes Studied with Label-Free High-Throughput Wide-Field Second-Harmonic Microscopy.

The interaction of oils and lipids is relevant for membrane biochemistry since the cell uses bilayer membranes, lipid droplets, and oily substances in its metabolic cycle. In addition, a variety of model lipid membrane systems, such as freestanding horizontal membranes and droplet interface bilayers, are made using oil to facilitate membrane monolayer apposition. We characterize the behavior of excess oil inside horizontal freestanding lipid bilayers using different oils, focusing on hexadecane and squalene. Using a combination of second-harmonic (SH) and white-light imaging, we measure how oil redistributes within the membrane bilayer after formation. SH imaging shows that squalene forms a wider annulus compared with hexadecane, suggesting that there is a higher quantity of squalene remaining in the bilayer compared with hexadecane. Excess oil droplets that appear right after membrane formation are tracked with white-light microscopy. Hexadecane droplets move directionally to the edge of the membrane with diffusion constants similar to those of single lipids, whereas squalene oil droplets move randomly with lower diffusion speeds similar to lipid condensed domains and remain trapped in the center of the bilayer for ∼1-3 h. We discuss the observed differences in terms of different coupling mechanisms between the oil and lipid molecules induced by the different chemical structures of the oils.

Label-free and charge-sensitive dynamic imaging of lipid membrane hydration on millisecond time scales.

Biological membranes are highly dynamic and complex lipid bilayers, responsible for the fate of living cells. To achieve this function, the hydrating environment is crucial. However, membrane imaging typically neglects water, focusing on the insertion of probes, resonant responses of lipids, or the hydrophobic core. Owing to a recent improvement of second-harmonic (SH) imaging throughput by three orders of magnitude, we show here that we can use SH microscopy to follow membrane hydration of freestanding lipid bilayers on millisecond time scales. Instead of using the UV/VIS resonant response of specific membrane-inserted fluorophores to record static SH images over time scales of >1,000 s, we SH imaged symmetric and asymmetric lipid membranes, while varying the ionic strength and pH of the adjacent solutions. We show that the nonresonant SH response of water molecules aligned by charge-dipole interactions with charged lipids can be used as a label-free probe of membrane structure and dynamics. Lipid domain diffusion is imaged label-free by means of the hydration of charged domains. The orientational ordering of water is used to construct electrostatic membrane potential maps. The average membrane potential depends quadratically on an applied external bias, which is modeled by nonlinear optical theory. Spatiotemporal fluctuations on the order of 100-mV changes in the membrane potential are seen. These changes imply that membranes are very dynamic, not only in their structure but also in their membrane potential landscape. This may have important consequences for membrane function, mechanical stability, and protein/pore distributions.

High throughput second harmonic imaging for label-free biological applications.

Second harmonic generation (SHG) is inherently sensitive to the absence of spatial centrosymmetry, which can render it intrinsically sensitive to interfacial processes, chemical changes and electrochemical responses. Here, we seek to improve the imaging throughput of SHG microscopy by using a wide-field imaging scheme in combination with a medium-range repetition rate amplified near infrared femtosecond laser source and gated detection. The imaging throughput of this configuration is tested by measuring the optical image contrast for different image acquisition times of BaTiO3 nanoparticles in two different wide-field setups and one commercial point-scanning configuration. We find that the second harmonic imaging throughput is improved by 2-3 orders of magnitude compared to point-scan imaging. Capitalizing on this result, we perform low fluence imaging of (parts of) living mammalian neurons in culture.