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.

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.

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. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu2+, Ca2+, Ba2+, Mg2+) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.

Far-field fluorescence spectromicroscopy of single quantum emitters (SQEs) (single molecules, quantum dots, color centers in crystals) is an actively developing field of modern photonics, which is in widespread demand in various applications in physics, chemistry, material sciences, life sciences, and quantum technologies. In this paper, we present a description of a multifunctional experimental setup which was developed in recent years at the Institute for Spectroscopy of the Russian Academy of Sciences. It allows measuring optical spectra and fluorescence images of SQEs, as well as their temporal behavior and luminescence kinetics, in a broad range of temperatures (from cryogenic to ambient). It is shown that the spatial coordinates of SQEs can be reconstructed with subdiffractional accuracy (up to a few angstroms). Some examples of the developed methods for multiparameter superresolution microscopy (nanoscopy) of materials and nanostructures are presented.