Significance: Lipid bilayer membranes are responsible for compartmentalization, signaling, transport, and flow of charge in living cells. Membranes self-assemble in aqueous solutions. Without a hydrating environment, membranes cannot exist. It is therefore surprising to note that the hydrating water is neglected in most membrane-related studies. We imaged membrane-bound oriented water by means of label-free second harmonic microscopy. We tracked, on millisecond time scales, membrane domain diffusion of condensed charged lipid domains, domain structure, and the spatial distribution of charge. Real-time electrostatic membrane potential maps were constructed using nonlinear optical theory. The spatiotemporal fluctuation in the membrane potential is surprisingly large and reveals the importance of charge fluctuations on membranes.

Water transport across lipid membranes is fundamental to all forms of life and plays a major role in health and disease. However, not only typical water facilitators like aquaporins facilitate water flux, but also transporters, ion channels or receptors represent potent water pathways. The efforts directed towards a mechanistic understanding of water conductivity determinants in transmembrane proteins, the development of water flow inhibitors, and the creation of biomimetic membranes with incorporated membrane proteins or artificial water channels depend on reliable and accurate ways of quantifying water permeabilities P_f. A conventional method is to subject vesicles to an osmotic gradient in a stopped-flow device: Fast recordings of scattered light intensity are converted into the time course of vesicle volume change. Even though an analytical solution accurately acquiring P_f from scattered light intensities exists, approximations potentially misjudging P_f by orders of magnitude are used. By means of computational and experimental data we point out that erroneous results such as that the single channel water permeability p_f depends on the osmotic gradient are direct results of such approximations. Finally, we propose an empirical solution of which calculated permeability values closely match those calculated with the analytical solution in the relevant range of parameters.

Vitamin C (VC)—a collective term for the different oxidation and protonation forms of ascorbic acid (AscH)—is an essential micronutrient that serves as (i) a potent antioxidant and (ii) a cofactor of a manifold of enzymatic processes. Its role in health is related to redox balance maintenance, which is altered in diseases such as obesity, cancer, neurodegenerative diseases, hypertension, and autoimmune diseases. Despite its importance, VC uptake has been poorly investigated. Available literature values for the passive membrane permeability P of lipid bilayers for AscH scatter by about 10 orders of magnitude. Here, we show by voltage clamp that $P^{-}$ of AscH’s anionic form (ascorbate ${\mathrm{Asc}}^{-}$ ) is negligible. To cross the membrane, ${\mathrm{Asc}}^{-}$ picks up a proton in the membrane vicinity and releases it on the other side of the membrane. This leads to a near-membrane pH drop that was visualized by scanning pH microelectrodes. The AscH concentration dependent pH profiles indicated $P=1.1\pm 0.1\times {10}^{-8}\mathrm{cm}/\mathrm{s}$ . Thus, AscH’s P is comparable to that of sorbitol and much lower than that of other weak acids like acetic acid or salicylic acid. The observation suggests that the capacity of the passive transcellular transport pathway across the lipid matrix does not suffice to ensure the required VC intake from the gastrointestinal tract.

Measurements of the unitary hydraulic conductivity of membrane channels, p_f, may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for p_f measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving p_f values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. p_f amounted to 2.4 ± 0.1 × 10⁻¹³ cm³ s⁻¹ for aquaporin‐1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on p_f on a single sample.