Second harmonic imaging of membrane potentials: Comparing water and chromophores as probes

The electrostatic potential of lipid membranes is an important metric to gain insight into the physiological state of cells. Second harmonic (SH) imaging has emerged as a powerful tool to perform in situ and in vivo electrostatic potential measurements, either using targeted probes or label-free by using water as a probe. In this work, we compare both modalities, by performing SH imaging on freestanding lipid membranes in aqueous solutions. Correlating the SH intensity to an applied external potential bias, the resonant voltage probe and the water response display distinctly different responses, which can be traced back to different nonlinear optical mechanisms. These mechanisms ensure that the water imaging provides a clean readout of the transmembrane potential, while the chromophore emission reports on the surface potential in the leaflet where it resides, together with differences in chromophore density, structure, and tilt angles. This means that, while both methods may show similar results, they cannot be compared directly. A combination of both methods is suggested as a useful strategy as the water response is accurate but currently limited to sub-second temporal resolution, while the resonant probe permits a sub-millisecond temporal resolution but is hampered by convoluted structural effects and chemical modification.