The interaction of divalent copper ions (Cu2+) with cell membranes is crucial for a variety of physiological processes of cells, such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies. Here, we use high-throughput wide-field second harmonic (SH) microscopy to study the interaction between Cu2+ and hydrated freestanding Montal-Muller lipid membranes. The symmetric lipid membranes are composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and either 1,2-diphytanoyl-sn-glycero-3-phosphate or 1,2-diphytanoyl-sn-glycero-3-phospho L-serine and are brought into contact with divalent Cu2+, which are added to one leaflet while maintaining the ionic strength balance. We observe transient domains of high SH intensity. In these areas, Cu2+ ions bind to the charged head groups, leading to charge neutralization on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane interface, which can be used to compute the interfacial membrane potential difference. We find that the domains of lipids with phosphatidic acid head groups display a higher interfacial membrane potential than those with phosphatidylserine head groups, which converts into higher dynamic electrostatic free energies and binding constants. (C) 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).