Resolving nanosecond kinetics of the optical membrane potential in pulsed electric fields

The primary bioeffect of pulsed electric fields (PEFs) is cell membrane charging, which determines a plethora of downstream events. While the kinetics of de- and hyperpolarization at the cathode- and anode-facing cell hemispheres should be the same, studies using pulsed laser strobe microscopy have reported asymmetrical charging in mammalian cells exposed to brief PEFs. To validate these anomalies, we measured charging kinetics with FluoVolt and Di-8-ANEPPS potentiometric dyes in spheroidal CHO cells exposed to 1-μs PEFs at 0-350 V/cm. Both dyes exhibited emission changes dependent on the electric field incidence angle, symmetrically at cathodic and anodic hemispheres. The changes scaled linearly with electric field strength up to at least 350 V/cm for Di-8-ANEPPS, but only to ∼200 V/cm for FluoVolt. Time constants measured from the optical membrane potential kinetics did not differ between the dyes, between cathodic and anodic cell poles, or between membrane charging and relaxation. The time constants followed the theoretical dependence on cell radius for 1.6 μF/cm² specific membrane capacitance and 0.4 S/m intracellular conductance. No charging anomalies were observed. Both dyes proved suitable for characterizing nanosecond charging kinetics, with FluoVolt being more sensitive for low-noise readings, while Di-8-ANEPPS allowed for dynamic measurements within a broader range.