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Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

Nanosecond bipolar pulse cancellation, a recently discovered phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (>100 μs) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results.

Our group recently published direct observation of membrane charging in FluoVolt labeled CHO-K1 cells by nanosecond electrical pulses using a streak camera. Using this technique, called Streak Camera Microscopy (SCM), we imaged the membrane charging dynamics in giant unilamellar vesicles (GUVs) during AC exposures up to 6 MHz and compared these results to existing capacitive circuit models of membranes. This work shows further application of Streak Camera Microscopy for evaluation of high speed biological events.

Exposures to short-duration, strong electric field pulses have been utilized for stimulation, ablation, and the delivery of molecules into cells. Ultrashort, nanosecond duration pulses have shown unique benefits, but they require higher field strengths. One way to overcome this requirement is to use trains of nanosecond pulses with high repetition rates, up to the MHz range. Here we present a theoretical model to describe the effects of pulse trains on the plasma membrane and intracellular membranes modeled as resistively charged capacitors. We derive the induced membrane potential and the stimulation threshold as functions of pulse number, pulse duration, and repetition rate. This derivation provides a straightforward method to calculate the membrane charging time constant from experimental data. The derived excitation threshold agrees with nerve stimulation experiments, indicating that nanosecond pulses are not more effective than longer pulses in charging nerve fibers. The derived excitation threshold does not, however, correctly predict the nanosecond stimulation of cardiomyocytes. We show that a better agreement is possible if multiple charging time constants are considered. Finally, we expand the model to intracellular membranes and show that pulse trains do not lead to charge buildup, but can create significant oscillations of the intracellular membrane potential.

Imaging of fluorescent small molecule transport into electropermeabilized cells reveals polarized patterns of entry, which must reflect in some way the mechanisms of the migration of these molecules across the compromised membrane barrier. In some reports, transport occurs primarily across the areas of the membrane nearest the positive electrode (anode), but in others cathode-facing entry dominates. Here we compare YO-PRO-1, propidium, and calcein uptake into U-937 cells after nanosecond (6 ns) and microsecond (220 µs) electric pulse exposures. Each of the three dyes exhibits a different pattern. Calcein shows no preference for anode- or cathode-facing entry that is detectable with our measurement system. Immediately after a microsecond pulse, YO-PRO-1 and propidium enter the cell roughly equally from the positive and negative poles, but transport through the cathode-facing side dominates in less than 1 s. After nanosecond pulse permeabilization, YO-PRO-1 and propidium enter primarily on the anode-facing side of the cell. Electronic supplementary material The online version of this article (doi:10.1007/s00232-017-9962-1) contains supplementary material, which is available to authorized users.

Intense nanosecond pulsed electric field (nsPEF) is a novel modality for cell activation and nanoelectroporation. Applications of nsPEF in research and therapy are hindered by a high electric field requirement, typically from 1 to over 50 kV/cm to elicit any bioeffects. We show how this requirement can be overcome by engaging temporal summation when pulses are compressed into high-rate bursts (up to several MHz). This approach was tested for excitation of ventricular cardiomyocytes and peripheral nerve fibers; for membrane electroporation of cardiomyocytes, CHO, and HEK cells; and for killing EL-4 cells. MHz compression of nsPEF bursts (100–1000 pulses) enables excitation at only 0.01–0.15 kV/cm and electroporation already at 0.4–0.6 kV/cm. Clear separation of excitation and electroporation thresholds allows for multiple excitation cycles without membrane disruption. The efficiency of nsPEF bursts increases with the duty cycle (by increasing either pulse duration or repetition rate) and with increasing the total time “on” (by increasing either pulse duration or number). For some endpoints, the efficiency of nsPEF bursts matches a single “long” pulse whose amplitude and duration equal the time-average amplitude and duration of the bursts. For other endpoints this rule is not valid, presumably because of nsPEF-specific bioeffects and/or possible modification of targets already during the burst. MHz compression of nsPEF bursts is a universal and efficient way to lower excitation thresholds and facilitate electroporation.

Dosage control of reactive oxygen and nitrogen species (RONS) is critical to low-temperature plasma applications in cancer therapy. Production of RONS by atmospheric pressure, nonequilibrium plasmas in contact with liquid may be modulated via plasma conditions including plasma treatment time and pulse voltage and repetition frequency. In this study, a terephthalic acid-based probe was used to measure hydroxyl radicals [OH(aq)] in water exposed to plasma and to demonstrate that the OH(aq) concentration increases linearly with treatment time. Fluorometric measurements of hydrogen peroxide concentration in plasma-activated water show a linear relationship between the H2O2 production rate and the pulse repetition frequency of the plasma. In vitro plasma treatment of cancer cells shows that pancreatic (Pan02) and breast (4T1-Luc) cancer cells have different sensitivities to plasma exposure. The dependence of Pan02 cell viability on plasma treatment time or pulse voltage is nonlinear. The system described here for generation and delivery of reactive oxygen species from a nanosecond pulsed plasma jet represents a promising alternative approach to cancer therapy.

The detailed molecular mechanisms underlying the permeabilization of cell membranes by pulsed electric fields (electroporation) remain obscure despite decades of investigative effort. To advance beyond descriptive schematics to the development of robust, predictive models, empirical parameters in existing models must be replaced with physics- and biology-based terms anchored in experimental observations. We report here absolute values for the uptake of YO-PRO-1, a small-molecule fluorescent indicator of membrane integrity, into cells after a single electric pulse lasting only 6 ns. We correlate these measured values, based on fluorescence microphotometry of hundreds of individual cells, with a diffusion-based geometric analysis of pore-mediated transport and with molecular simulations of transport across electropores in a phospholipid bilayer. The results challenge the “drift and diffusion through a pore” model that dominates conventional explanatory schemes for the electroporative transfer of small molecules into cells and point to the necessity for a more complex model.

High-intensity nanosecond pulsed electric fields (nsPEFs) permeabilize cell membranes. Although progress has been made toward an understanding of the mechanism of nsPEF-induced membrane poration, the dependence of pore size and distribution on pulse duration, strength, number, and repetition rate remains poorly defined experimentally. In this paper, we characterize the size of nsPEF-induced pores in living cell membranes by isosmotically replacing the solutes in pulsing media with polyethylene glycols and sugars before exposing Jurkat T lymphoblasts to 5 ns, 10 MV/m electric pulses. Pore size was evaluated by analyzing cell volume changes resulting from the permeation of osmolytes through the plasma membrane. We find that pores created by 5 ns pulses have a diameter between 0.7 and 0.9 nm at pulse counts up to 100 with a repetition rate of 1 kHz. For larger number of pulses, either the pore diameter or the number of pores created, or both, increase with increasing pulse counts. But the prevention of cell swelling by PEG 1000 even after 2000 pulses suggests that 5 ns, 10 MV/m pulses cannot produce pores with a diameter larger than 1.9 nm.

Nanosecond pulsed helium plasma jets impinging on water produce hydroxyl radicals both in gas- and liquid-phase. In this study, the effects of pulse width on a repetitively pulsed plasma jet in contact with water are evaluated via OH(A–X) emission and OHaq production in water for various pulse widths ranging from 200 to 5000 ns. The maximal energy efficiency of OH(A–X) emission is obtained for pulse widths of 600–800 ns whereas the maximal efficiency of OHaq production is at 200 ns. Temporally-resolved emission spectroscopy shows that more than 40% of OH(A–X) emission is produced during the first 200 ns of the voltage pulse regardless of the pulse width. An equivalent circuit model of the plasma jet impinging on water is compiled to understand the charge transfer process, which is important for OHaq production via charge exchange reactions.

Excited by 5 ns, 8 kV voltage pulses, a 260 μm-diameter, 8 mm long helium plasma jet was generated with a single-electrode configuration in ambient air. Application of fast high voltage pulses (≥1012 V s−1) resulted in rapid acceleration of the microplasma plumes; within 5 ns the plume velocity reached 8 × 105 m/s, almost three times higher than that of the plasma jet generated with the pulsed voltage of the same amplitude but with a lower increase rate (1011 V s−1). Importantly, the ultrashort electric pulses were able to efficiently deposit energy in the plasma during the initiation process, which may be responsible for the rapid acceleration of the ionization wavefronts during the streamer onset, as well as efficient production of reactive plasma species including O(5P) and N2+(B2Σu+) via electron-induced processes. Emission spectral comparison between the plasma jets excited with 5 ns voltage pulses and with 140 ns voltage pulses showed enhanced O(5P) and N2+(B2Σu+) emission by the shorter pulses than the longer ones, while the vibrational and rotational temperature for both plasma jets are at 3000 K and 300 K, respectively.

High amplitude, nanosecond-duration, pulsed electric fields (nsPEFs) induce the formation of nanometer-diameter pores in cell membranes. These transient lipid electropores facilitate the transport of normally impermeant ions and small molecules across the membrane. Quantitative measurements of molecular transport through lipid electropores are essential for the validation of electroporation models and for optimizing the nsPEF dose for application-specific responses. We calibrated the fluorescence intensity of the membrane integrity marker dye YO-PRO-1 in dense cell lysates using a fast confocal microscope in order to quantify the time-dependent molecular influx of YO-PRO-1 into living cells after nsPEF exposure. Using this method we were able to measure the number of YO-PRO-1 molecules that enter U-937 human histiocytic lymphoma cells following electropermeabilization by a single 6 ns, 20 MV/m pulse.