Hydrated lipid bilayer membranes are crucial components of cells and organelles, serving as the outer boundary that separates the cellular components from the extracellular environment. Lipid membranes regulate their structures and functions by dynamically responding to external perturbations. However, the effect of such perturbations on a hydrated interface has not been well understood due to a lack of experimental techniques that can probe the complex structure of the membrane hydration. In this thesis, we present a novel approach to investigate the spatiotemporal evolution of the hydration structure of freestanding lipid membranes when exposed to external perturbations using wide-field second harmonic (SH) microscopy. We show the response of interfacial water can be SH imaged in a series of experiments involving the lipid membranes influenced by thermal flux, osmotic stress, ion binding, and ion permeation. We use this water response as a contrast mechanism to quantify the molecular interfacial structures and properties of the membranes. We start with SH imaging of the membrane hydration under various conditions, including changes in temperature of adjacent solutions to the membranes and lipid compositions. The hydrating water exhibits a nonrandom orientation, which arises from chemical interactions at membrane interfaces as well as charge-dipole interactions. We find the thermal gradient causes an asymmetrical expansion of inter-lipid spacing between inner and outer leaflets. This in turn polarizes the lipid membranes and decreases surface charge density. Next, we use SH microscopy and capacitance measurement to probe osmosis-induced hydration and polarization of the lipid membranes. We observe osmotic stress leads to an increase in membrane capacitance, which indicates the deformation of the membranes. SH imaging exhibits that membrane deformation gives rise to lipid-water structural anisotropy between leaflets, polarizing the lipid bilayer. By varying lipid compositions, we find the impact of osmosis on membrane hydration is greatly determined by the area per lipid. Following that, we demonstrate the electrostatic interactions of Cu2+ ions with water and negatively charged lipid membranes. The interaction induces the transient micron-sized domains of high SH intensity where Cu2+ ions bind to the charged head groups, neutralizing the charge on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane, enabling computation of the interfacial membrane potential difference. We find that lipids with the same charge but different chemical structures have different electrostatic potentials as well as binding constants when they are bound to Cu2+ ions. Lastly, we present a link between fluctuations in membrane potential and proton translocation. SH imaging shows that proton-membrane interaction causes potential fluctuations. Molecular dynamics simulations suggest these potential fluctuations impact the energetics of defect formation, lowering them to the level needed to allow for the transport of protons. We find a thin water needle is formed in a defect and allows translocation of the proton. SH imaging and conductivity measurement reveals that the rate of proton transport varies with the structure of the hydrophobic core in bilayer membranes. Combining experimental and computational results, we explain a mechanism for proton translocation via a water needle induced by the membrane potential.