Hydrated lipid bilayer membranes and their asymmetry play a fundamental role in living cells by maintaining and regulating concentration gradients between cells, their environment, and their compartments. They achieve this not only through various channels and transporters, but also by being inherently semi-permeable to various molecules, including water and ions. However, molecular information about passive ion transport, as well as the complex structure of membrane hydration remain elusive, mainly due to a lack of experimental methods that can access this information, and relate it to micro- and macroscopic membrane properties. In this work, we study the molecular structure of lipid bilayer membranes and ion transport across them with high throughput wide-field second harmonic (SH) microscopy by utilizing water as the contrast agent. We show that by detecting small amounts of asymmetry in the structure of interfacial water, it is possible to measure the distribution of charged species across the membrane with high sensitivity. We apply this surface-selective technique to measure ion-lipid-water interactions, track ion transport, and quantify electro-chemical surface properties at the interfaces of lipid bilayer membranes in the form of giant unilamellar vesicles (GUVs). We start by improving the throughput of wide field SH microscopy in order to probe low-asymmetry interfaces with high contrast in a label-free manner. To do this, we design a custom optical parametric amplifier (OPA) with a tunable output in the 670-1000 nm range, up to a 1 MHz repetition rate, and an ultra-short 23 fs pulse duration. We then experimentally demonstrate the achieved throughput improvement. Next, we establish a way to probe interfacial hydration of GUVs. We quantify the surface properties of vesicles composed of different ratios of zwitterionic and anionic lipids, and show that only a few percent of anionic headgroups are ionized. We also observe spatial and temporal fluctuations in surface properties, and demonstrate that these fluctuations are universally found in lipid bilayer systems. Following that, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. Molecular dynamics simulations reveal that these fluctuations 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 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. Then, we focus on unassisted Ca2+ translocation in more detail. We vary the hydrophobic core of bilayer membranes and observe different types of behavior in high throughput wide-field SH images. Ca2+ translocation is observed through mono-unsaturated membranes, significantly reduced upon adding cholesterol, and completely inhibited for branched and poly-unsaturated membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion induced transient pores, which require non-equilibrium membrane restructuring. This results in different transport rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.