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Title:
Non-destructive real-time imaging of membrane charging and relaxation in cells subjected to brief electric field pulses
Authors:
Semenov, Iurii - 1
Kim, Vitalii - 1
Bixler, Joel - 2
Kiester, Allen - 2
Ibey, Bennett - 2
Pakhomov, Andrei G.- 1
1 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA
2 Air Force Research Laboratory, JBSA Fort Sam Houston, TX
Abstract:
Most, if not all, bioeffects of electric pulses are determined by the charging and relaxation of the cell plasma membrane. Therefore, measuring these processes' exact kinetics is critical for the mechanistic explanation of most bioelectric phenomena, including cell excitation and electroporation, bipolar cancellation, and facilitation of bioeffects by high-frequency pulse trains. We have employed strobe photography with a fast voltage-sensitive membrane dye FluoVolt for direct and non-destructive imaging of membrane charging and relaxation kinetics [1-3]. In brief, the dye was excited by ultrashort laser pulses (~8 ns) delivered with a variable delay with respect to a nano- or microsecond electric pulse (nsEP and µsEP) exposure of the cell loaded with the dye. The delay was varied in 50-or 100-ns increments or decrements so that the laser flashed at different times before, during, and after EP. The camera shutter was opened beforehand and captured a single fluorescence image at the moment of the laser flash. We delivered a total of up 50- to 200- pulses to acquire the respective number of images and build the time kinetics of plasma membrane charging and discharging.
The method was validated in CHO cells. The same number of pulses and laser flashes were delivered at a constant time delay. It showed that the response of the dye remained unchanged, indicating no damage to the observed cell. We showed that the fluctuations in the excitation light intensity could be offset
entirely by the normalization of the data to the integral of the whole-cell fluorescence (which remained unaffected by the external electric field below the electroporation threshold). Kinetic measurements
were the same in trials where we increase the time delay between the EP and the laser flash and when we decrease it. Also, kinetic measurements for a given cell were the same no matter which side of it was facing the anode or cathode. Measurements for different selections of the region of interest (ROI) on
the cell membrane were compared to show that ROI size does not change plasma membrane parameters such as membrane time constant if it is located at the cathode or anode side of the cell.
A novel and unexpected observation made was a striking difference in the cell membrane's charging and discharging time constants. For example, for 1-µs pulses at 0.34 kV/cm, the average charging time constant was 0.26 +/-0.01 µs, and the discharging one was 0.56 +/- 0.06 µs (p<0.01). The membrane discharge constant depended much more on the cell’s size than the membrane charge constant.
Possible underlying mechanisms and implications of this observation will be discussed.
Keywords:
Refs:
1. Kim, V.; Semenov, I.; Kiester, A. S.; Keppler, M. A.; Ibey, B. L.; Bixler, J. N.; Pakhomov, A. G.,
Action spectra and mechanisms of (in) efficiency of bipolar electric pulses at electroporation.
Bioelectrochemistry 2023, 149, 108319.
2. Gudvangen, E.; Mangalanathan, U.; Semenov, I.; Kiester, A. S.; Keppler, M. A.; Ibey, B. L.; Bixler,
J. N.; Pakhomov, A. G., Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating
Damage to Smooth Muscle: An In Vitro Study. Int J Mol Sci 2023, 24, (3).
3. Kiester, A. S.; Ibey, B. L.; Coker, Z. N.; Pakhomov, A. G.; Bixler, J. N., Strobe photography mapping
of cell membrane potential with nanosecond resolution. Bioelectrochemistry 2021, 142, 107929.
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