Plasma Membrane Charging and Electropermeabilization Measured Using Strobe Fluorescence Microscopy
Document Type
Abstract
Publication Date
11-7-2024
DOI
10.25776/edk0-d502
Abstract
Chinese Hamster Ovary (CHO) cells loaded with the voltage-sensitive FluoVolt dye were exposed to 1-µs electric pulses at field strengths both below and above the electropermeabilization threshold. The dye was excited by ~8-ns laser flashes delivered at varied time intervals relative to the electric pulse. The delays were adjusted in 50-ns increments or decrements, allowing for the collection of cell images at different times during the charging and relaxation phases of the induced transmembrane potential (TMP).
Cells were exposed to field strengths ranging from 0.09 to 0.48 kV/cm to analyze the dependence of FluoVolt fluorescence on plasma membrane (PM) charge and cell diameter. The resulting curve displayed a sigmoidal shape, with linearity within ±30% of the resting level. The experimental data correlated with theoretical predictions, showing a linear range corresponding to a ±320 mV change in TMP. Outside of this range, the FluoVolt signal lost linearity with respect to the increasing electric field, likely due to PM electropermeabilization. However, electropermeabilization at these field strengths was not detected using standard methods, such as YO-PRO or Ca2+ uptake assays.
The induced TMP was highest at the cell poles facing the electrodes and gradually decreased toward the cell equator. This TMP distribution aligned with theoretical predictions for most cells exposed to 0.09-0.15 kV/cm. However, larger cells exposed to field strengths of ≥0.25 kV/cm exhibited a lower induced TMP at the poles than predicted.
Application of a single pulse or pair of consecutive pulses at various intervals (ranging from 1 to 250 µs) showed that the PM could be charged again by the second pulse in the pair to the same level as it was charged by the single pulse of the same amplitude even if the 1st pulse was well above electropermeabilization threshold (in fact, in a separate set of experiments, Ca2+ uptake was observed upon exposure to a pulse of the same amplitude and duration). These findings suggest that the PM recovery time after moderate electropermeabilization may be shorter than 1 µs.
In conclusion, our data demonstrate that strobe microscopy is a sensitive tool capable of detecting PM electropermeabilization at thresholds lower than those detected by conventional methods. This technique also provides valuable insights into the dynamics of charging and electropermeabilization, as well as TMP distribution across the PM during pulse exposure.
Repository Citation
Semenov, Iurii; Bixler, Joel; Ibey, Bennett; and Pakhomov, Andrei, "Plasma Membrane Charging and Electropermeabilization Measured Using Strobe Fluorescence Microscopy" (2024). 2024 Frank Reidy Research Center for Bioelectrics Retreat. 5.
https://digitalcommons.odu.edu/bioelectrics-2024retreat/5
