Sub-microsecond optical measurements of cell membrane charging and lesioning by pulsed electric fields

Membrane charging and permeabilization by 1- and 0.3-μs pulsed electric fields (PEFs) were visualized and quantified in spheroidal CHO cells using pulsed laser fluorescence microscopy. FluoVolt and Di-8-ANEPPS dyes changed emission by 26% and 9.1% per 100 mV induced membrane potential (IMP). FluoVolt saturated at +255 mV and - 320 mV IMP, whereas Di-8-ANEPPS responded from <-800 to >+550 mV. Measured IMP matched theory and followed cosine dependence on incidence angle, reaching ±500 mV at cell poles. Charging weakened once IMP exceeded ∼500 mV (at 570 and 1000 V/cm for 1- and 0.3-μs PEFs), indicating electroporation-driven conductivity increase. Stronger PEFs further increased conductivity, especially at the cathodic pole, limiting IMP to ∼±650 mV (-780 mV with 1 mM Gd³⁺). Electroporation was confirmed by Ca²⁺ entry with 1-μs PEFs >650 V/cm. Electroporation-induced effective conductivity at cell poles fell ∼10-fold within 6 μs after 1-μs PEFs; recovery was complete after ≤780 V/cm, but residual conductivity persisted after stronger PEFs. Pulsed laser IMP imaging proved uniquely capable of resolving early electroporation events despite uncertainties of dye calibration at high IMPs and potential cumulative effects of repeated electroporating pulses.