On the mechanism and voltage sensitivity of voltage-sensitive second harmonic probes: A case study of FM4-64

Voltage-sensitive dyes are frequently used to measure electrostatic potentials across (cell) membranes. Their use is based on the observed linear correlation between the second harmonic (SH) emission and an applied transmembrane potential. However, this intensity-to-potential conversion implicitly assumes a single, fixed dye orientation and a constant surface number density in the membrane, both taken to be independent of the local molecular environment. Here, we investigate whether these assumptions hold by imaging the SH response of a voltage-sensitive dye (FM4-64) in giant unilamellar vesicles. Using polarimetric SH microscopy, we determine changes in the orientational distribution parameter D and surface number density of FM4-64. We find that the orientational distribution of FM4-64 is influenced by the type of lipid and the charged state of the membrane. Zwitterionic saturated and unsaturated lipids display different orientational distributions of FM4-64, and the charge density of charged membranes also influences the orientational distribution, with higher charge opening the possibility of broader orientational distributions. The addition of Ca²⁺ ions in the solution leads to a change in the number of adsorbed probe molecules, which can be quantified for zwitterionic lipids. Because variations in the orientational distribution and surface number density due to different lipid compositions and ionic environment result in widely different SH responses, these factors must be accounted for when interpreting SH signals from voltage-sensitive probes.