The transition from field emission to collisional space-charge limited current with nonzero initial velocity.

Multiple electron emission mechanisms often contribute in electron devices, motivating theoretical studies characterizing the transitions between them. Previous studies unified thermionic and field emission, defined by the Richardson-Laue-Dushman (RLD) and Fowler-Nordheim (FN) equations, respectively, with the Child-Langmuir (CL) law for vacuum space-charge limited current (SCLC); another study unified FN and CL with the Mott-Gurney (MG) law for collisional SCLC. However, thermionic emission, which introduces a nonzero injection velocity, may also occur in gas, motivating this analysis to unify RLD, FN, CL, and MG. We exactly calculate the current density as a function of applied voltage over a range of injection velocity (i.e., temperature), mobility, and gap distance. This exact solution approaches RLD, FN, and generalized CL (GCL) and MG (GMG) for nonzero injection velocity under appropriate limits. For nonzero initial velocity, GMG approaches zero for sufficiently small applied voltage and mobility, making these gaps always space-charge limited by either GMG at low voltage or GCL at high voltage. The third-order nexus between FN, GMG, and GCL changes negligibly from the zero initial velocity calculation over ten orders of magnitude of applied voltage. These results provide a closed form solution for GMG and guidance on thermionic emission in a collisional gap.

Selective Electroporation of Tumor Cells Under AC Radiofrequency Stimulation - A Numerical Study.

Self-consistent evaluations of membrane electroporation along with local heating in single spherical cells arising from external AC radiofrequency electrical stimulation have been carried out. The present numerical study seeks to determine whether healthy and malignant cells exhibit separate electroporative responses with regards to operating frequency. It is shown that cells of Burkitt's lymphoma would respond to frequencies >4.5 MHz, while normal B-cells would have negligible porative effects in that higher frequency range. Similarly, a frequency separation between the response of healthy T-cells and malignant species is predicted with a threshold of about 4 MHz for cancer cells. The present simulation technique is general and so would be able to ascertain the beneficial frequency range for different cell types. The demonstration of higher frequencies to induce poration in malignant cells, while having minimal affecting healthy ones, suggests the possibility of selective electrical targeting for tumor treatments and protocols. It also opens the doorway for tabulating selectivity enhancement regimes as a guide for parameter selection towards more effective treatments while minimizing deleterious effects on healthy cells and tissues.

Infrared Laser-Based Single Cell Permeabilization by Plasma Membrane Temperature Gradients.

Single cell microinjection provides precise tuning of the volume and timing of delivery into the treated cells; however, it also introduces workflow complexity that requires highly skilled operators and specialized equipment. Laser-based microinjection provides an alternative method for targeting a single cell using a common laser and a workflow that may be readily standardized. This paper presents experiments using a 1550 nm, 100 fs pulse duration laser with a repetition rate of 20 ns for laser-based microinjection and calculations of the hypothesized physical mechanism responsible for the experimentally observed permeabilization. Chinese Hamster Ovarian (CHO) cells exposed to this laser underwent propidium iodide uptake, demonstrating the potential for selective cell permeabilization. The agreement between the experimental conditions and the electropermeabilization threshold based on estimated changes in the transmembrane potential induced by a laser-induced plasma membrane temperature gradient, even without accounting for enhancement due to traditional electroporation, strengthens the hypothesis of this mechanism for the experimental observations. Compared to standard 800 nm lasers, 1550 nm fs lasers may ultimately provide a lower cost microinjection method that readily interfaces with a microscope and is agnostic to operator skill, while inducing fewer deleterious effects (e.g., temperature rise, shockwaves, and cavitation bubbles).

Space-charge-limited current density for nonplanar diodes with monoenergetic emission using Lie-point symmetries.

Understanding space-charge-limited current density (SCLCD) is fundamentally and practically important for characterizing many high-power and high-current vacuum devices. Despite this, no analytic equations for SCLCD with nonzero monoenergetic initial velocity have been derived for nonplanar diodes from first principles. Obtaining analytic equations for SCLCD for nonplanar geometries is often complicated by the nonlinearity of the problem and over constrained boundary conditions. In this Letter, we use the canonical coordinates obtained by identifying Lie-point symmetries to linearize the governing differential equations to derive SCLCD for any orthogonal diode. Using this method, we derive exact analytic equations for SCLCD with a monoenergetic injection velocity for one-dimensional cylindrical, spherical, tip-to-tip (t-t), and tip-to-plate (t-p) diodes. We specifically demonstrate that the correction factor from zero initial velocity to monoenergetic emission depends only on the initial kinetic and electric potential energies and not on the diode geometry and that SCLCD is universal when plotted as a function of the canonical gap size. We also show that SCLCD for a t-p diode is a factor of four larger than a t-t diode independent of injection velocity. The results reduce to previously derived results for zero initial velocity using variational calculus and conformal mapping.

Nonlinear transmission line implemented as a combined pulse forming line and high-power microwave source.

Nonlinear transmission lines (NLTLs) are typically driven by pulse forming lines (PFLs) or Marx generators to generate high repetition rate, high power microwaves (HPMs) with fewer auxiliary systems than conventional sources. This paper reports the development of an even more compact HPM system that utilizes a composite-based hybrid NLTL as the PFL and HPM generator in a single device. We designed the following three different combinations of nickel zinc ferrite (NZF) and barium strontium titanate (BST) inclusion volume loads in a polydimethylsiloxane host material to provide magnetic field dependent permeability and electric field dependent permittivity, respectively: 25% NZF, 10% BST/15% NZF, and 15% BST/10% NZF. By constructing the NLTL in a coaxial geometry, this device uses the capacitance and length of the NLTL to generate a fast rise-time high voltage pulse with microwave oscillations that occurred both during and after the pulse after exceeding a threshold charging voltage. The output frequency of the NLTLs ranged from 950 MHz to 2.2 GHz during the pulse for all volume loadings and was 1 GHz after the pulse for the 10% BST/15% NZF and 15% BST/10% NZF volume loadings. The oscillations generated after the pulse were much higher in amplitude and achieved 160 kW at a 15 kV charging voltage for the 15% BST/10% NZF composite-based NLTL.

Electrical stimulation of whole blood for growth factor release and potential clinical implications.

Clinicians have increasingly applied platelet-rich plasma (PRP) for wound healing treatments. Topical treatments commonly require biochemical agents such as bovine thrombin to activate PRP ex vivo for clotting and growth factor release to facilitate healing upon application to the wound of interest. Recent studies have explored electrical stimulation as an alternative to bovine thrombin for PRP activation due to the former's cost, workflow complexity and potentially significant side effects; however, both approaches require separating the PRP from whole blood (WB) prior to activation. Eliminating the separation (typically centrifugation) step would reduce the cost and duration of the clinical procedure, which may be critical in trauma and surgical applications. We hypothesize that electric pulses (EPs) can release growth factors from WB, as they do from PRP, without requiring centrifugation of WB into PRP. A pilot study for two donors demonstrates the potential for EP stimulated growth factor release from WB. This motivates future experiments assessing EP parameter optimization for WB activation and in vivo studies to determine the clinical benefits for topical treatments and, especially, for injections in orthopedic applications that already utilize non-treated/non-activated WB.

Nanosecond electric pulses rapidly enhance the inactivation of Gram-negative bacteria using Gram-positive antibiotics.

Physically disrupting microorganism membranes to enable antibiotics to overcome resistance mechanisms that inhibit or excrete antibiotics has great potential for reducing antibiotic doses and rendering resistance mechanisms inert. We demonstrate the synergistic inactivation of a Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria by combining 222 30 kV/cm electric pulses (EPs) or 500 20 kV/cm EPs with 300-ns EP duration with various antibiotics with different mechanisms of action is demonstrated. Doses of antibiotics that produced no inactivation in 10 min of exposure in solution with bacteria induced several log reductions under the influence of nanosecond EPs. Combining 2 µg/L or 20 µg/mL of rifampicin with the 30 kV/cm EPs enhanced Staphylococcus aureus inactivation compared with EPs alone, while only a few of the other combinations demonstrated improvement. Combining 2 µg/L or 20 µg/mL of mupirocin or rifampicin with either EP train enhanced E. coli inactivation compared with EPs alone. Combining 2 µg/L or 20 µg/mL of erythromycin or vancomycin with the 30 kV/cm EPs enhanced E. coli inactivation compared with EPs alone. These results indicate that EPs can make Gram-positive antibiotics efficient for inactivating Gram-negative bacteria with future studies required to optimize EP parameters for other antibiotics and Gram-negative bacteria.

Pulsed electric field inactivation of microorganisms: from fundamental biophysics to synergistic treatments.

The growth of antibiotic resistant microorganisms and the increasing demand for nonthermal antimicrobial treatment in the food and beverage industry motivates research into alternative inactivation methods. Pulsed electric fields (PEFs) provide an athermal method for inactivating microorganisms by creating nanometer-sized membrane pores in microorganisms, inducing cell death when the PEF duration and intensity are sufficient such that the pores cannot reseal after the PEFs through a process referred to as irreversible electroporation. While PEF inactivation has been studied for several decades, recent studies have focused on extending the technique to various liquids in the food industry and optimizing microorganism inactivation while minimizing adverse effects to the treated sample. This minireview will assess the biophysical mechanisms and theory of PEF-induced cellular interactions and summarize recent advances in applying this technology for microorganism inactivation alone and synergistically in combination with other technologies, including temperature, pressure, natural ingredients, and pharmaceuticals.

Nanosecond pulsed electric field induced proliferation and differentiation of osteoblasts and myoblasts.

Low-intensity electric fields can induce changes in cell differentiation and cytoskeletal stresses that facilitate manipulation of osteoblasts and mesenchymal stem cells; however, the application times (tens of minutes) are of the order of physiological mechanisms, which can complicate treatment consistency. Intense nanosecond pulsed electric fields (nsPEFs) can overcome these challenges by inducing similar stresses on shorter timescales while additionally inducing plasma membrane nanoporation, ion transport and intracellular structure manipulation. This paper shows that treating myoblasts and osteoblasts with five 300 ns PEFs with intensities from 1.5 to 25 kV cm-1 increased proliferation and differentiation. While nsPEFs above 5 kV cm-1 decreased myoblast population growth, 10 and 20 kV cm-1 trains increased myoblast population by approximately fivefold 48 h after exposure when all cell densities were set to the same level after exposure. Three trials of the PEF-treated osteoblasts showed that PEF trains between 2.5 and 10 kV cm-1 induced the greatest population growth compared to the control 48 h after treatment. Trains of nsPEFs between 1.5 and 5 kV cm-1 induced the most nodule formation in osteoblasts, indicating bone formation. These results demonstrate the potential utility for nsPEFs to rapidly modulate stem cells for proliferation and differentiation and motivate future experiments to optimize PEF parameters for in vivo applications.

The Transition to Paschen's Law for Microscale Gas Breakdown at Subatmospheric Pressure.

The decrease in electronic device size necessitates greater understanding of gas breakdown and electron emission at microscale to optimize performance. While traditional breakdown theory using Paschen's law (PL), driven by Townsend avalanche, fails for gap distance d [Formula: see text] 15 µm, recent studies have derived analytic equations for breakdown voltage when field emission and Townsend avalanche drive breakdown. This study derives a new analytic equation that predicts breakdown voltage VB within 4% of the exact numerical results of a previously derived theory and new experimental results at subatmospheric pressure for gap distances from 1-25 µm. At atmospheric pressure, VB transitions to PL near the product of pressure and gap distance, pd, corresponding to the Paschen minimum; at lower pressures, the transition to PL occurs to the left of the minimum. We further show that the work function plays a major role in determining which side of the Paschen minimum VB transitions to PL as pressure approaches atmospheric pressure while field enhancement and the secondary emission coefficient play smaller roles. These results indicate that appropriate combinations of these parameters cause VB to transition to PL to the left of the Paschen minimum, which would yield an extended plateau similar to some microscale gas breakdown experimental observations.

Using extracellular calcium concentration and electric pulse conditions to tune platelet-rich plasma growth factor release and clotting.

Platelet-rich plasma (PRP) is an emerging autologous biologic method for wound healing. Clinicians apply PRP either topically (where it is activated ex-vivo before treatment by adding an external agent to trigger clotting and the release of growth factors that facilitate wound healing) or through injection (where it is activated in vivo at the injury site with no prior activation before injection). Because topical PRP activation typically utilizes bovine thrombin, which has significant potential side effects and high costs, recent studies have assessed the efficacy of combining extracellular calcium (EC) and electric pulses (EPs) to activate PRP. The potential to apply this novel technique to PRP both topically and internally via injection raises the question about the ability to tune the clotting time and growth factor release for a given application. While previous studies have assessed the impact of applying EPs of various durations either directly (conductive coupling) or indirectly (capacitive coupling) to PRP containing EC, no studies have assessed the tunability of this activation based on modifying EP parameters, EP delivery method (conductive or capacitive coupling), and the EC concentration. We hypothesize that tuning these parameters will modify intracellular calcium uptake to permit the control of growth factor release and clotting time, which are critical for optimizing PRP for either topical or internal clinical applications. A pilot study for a single donor demonstrates the potential for tunability as a function of the intensity of membrane manipulation and calcium concentration, which facilitate the increase of cytosolic calcium. This motivates future studies assessing EC and EP optimization and in vivo studies to determine the overall efficacy of this tunability for wound healing.

Synergistic bacterial inactivation by combining antibiotics with nanosecond electric pulses.

Antibiotic resistance mechanisms render current antibiotics ineffective, requiring higher concentrations of existing drugs or the development of more powerful drugs for infection treatment. This study demonstrates the synergistic inactivation of a gram-positive (Staphylococcus aureus) and a gram-negative (Escherichia coli) bacteria by combining either tobramycin or rifampicin with 300-ns electric pulses (EPs). For EPs depositing the same total energy density into the sample with no drug, higher electric fields induced greater inactivation, indicating a threshold for irreversible electroporation at these fields and membrane recovery in between lower intensity EPs. Synergistic inactivation generally increased with increasing drug concentration up to 20 µg/mL compared to strictly EP treatment. Combining even 1/20 of the clinical dose of tobramycin with a train of EPs induced between 2.5 and 3.5 log inactivation after only 10 min of exposure compared to hours to induce inactivation with a clinical dose with no EPs. Similarly, combining a train of EPs with a clinically relevant dose of rifampicin induced 7 to 9 log inactivation over the same time of exposure. These results indicate the promise of combining EPs with antibiotics to rapidly inactivate antibiotic-resistant bacteria in localized treatment areas.

Fabrication and characterization of implantable flushable electrodes for electric field-mediated drug delivery in a brain tissue-mimic agarose gel.

Every forty minutes, one person dies in the USA due to glioblastoma multiforme; a deadly form of brain cancer with an average five-year survival rate less than 3%. The current standard of care for treatment involves surgical resection of the accessible tumor followed by radiation therapy and concomitant chemotherapy. Despite their potency, delivering chemotherapeutic agents to the brain is limited by the highly selective blood-brain barrier, which prevents molecules >500 Da from reaching the brain. Other techniques, such as convection-enhanced delivery, controlled release by drug-loaded wafers or intracerebroventricular infusion have limited clinical utility due to unpredictable targeting and volume of drug distribution. We introduce a novel drug delivery technique that can use direct current electric fields to deliver charged chemotherapeutics to the site of brain parenchyma after tumor resection. We fabricate and characterize an implantable drug delivery system using flushable electrodes to deliver the charged chemotherapeutic or doxorubicin (+1) in a brain tissue-mimic agarose gel (0.2% w/v) model by electrophoresis. The optimized capillary-embedded electrode system exhibited a sustained movement of charged doxorubicin through nearly 3.5 mm in four hours, a distance for achieving effective intratumoral concentrations.

Design, characterization and experimental validation of a compact, flexible pulsed power architecture for ex vivo platelet activation.

Electric pulses can induce various changes in cell dynamics and properties depending upon pulse parameters; however, pulsed power generators for in vitro and ex vivo applications may have little to no flexibility in changing the pulse duration, rise- and fall-times, or pulse shape. We outline a compact pulsed power architecture that operates from hundreds of nanoseconds (with the potential for modification to tens of nanoseconds) to tens of microseconds by modifying a Marx topology via controlling switch sequences and voltages into each capacitor stage. We demonstrate that this device can deliver pulses to both low conductivity buffers, like standard pulsed power supplies used for electroporation, and higher conductivity solutions, such as blood and platelet rich plasma. We further test the effectiveness of this pulse generator for biomedical applications by successfully activating platelets ex vivo with 400 ns and 600 ns electric pulses. This novel bioelectrics platform may provide researchers with unprecedented flexibility to explore a wide range of pulse parameters that may induce phenomena ranging from intracellular to plasma membrane manipulation.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

BACKGROUND: Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. AIMS: To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. METHODS: PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. RESULTS: PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. CONCLUSIONS: PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

Plasma membrane temperature gradients and multiple cell permeabilization induced by low peak power density femtosecond lasers.

Calculations indicate that selectively heating the extracellular media induces membrane temperature gradients that combine with electric fields and a temperature-induced reduction in the electropermeabilization threshold to potentially facilitate exogenous molecular delivery. Experiments by a wide-field, pulsed femtosecond laser with peak power density far below typical single cell optical delivery systems confirmed this hypothesis. Operating this laser in continuous wave mode at the same average power permeabilized many fewer cells, suggesting that bulk heating alone is insufficient and temperature gradients are crucial for permeabilization. This work suggests promising opportunities for a high throughput, low cost, contactless method for laser mediated exogenous molecule delivery without the complex optics of typical single cell optoinjection, for potential integration into microscope imaging and microfluidic systems.

Platelet activation using electric pulse stimulation: growth factor profile and clinical implications.

BACKGROUND: Autologous platelet gel therapy using platelet-rich plasma has emerged as a promising alternative for chronic wound healing, hemostasis, and wound infection control. A critical step for this therapeutic approach is platelet activation, typically performed using bovine thrombin (BT) and calcium chloride. However, exposure of humans to BT can stimulate antibody formation, potentially resulting in severe hemorrhagic or thrombotic complications. Electric pulse stimulation using nanosecond PEFs (pulse electric fields) is an alternative, nonbiochemical platelet activation method, thereby avoiding exposure to xenogeneic thrombin and associated risks. METHODS: In this study, we identified specific requirements for a clinically relevant activator instrument by dynamically measuring current, voltage, and electric impedance for platelet-rich plasma samples. From these samples, we investigated the profile of growth factors released from human platelets with electric pulse stimulation versus BT, specifically platelet-derived growth factor, transforming growth factor ß, and epidermal growth factor, using commercial enzyme-linked immunosorbent assay kits. RESULTS: Electric pulse stimulation triggers growth factor release from platelet α-granules at the same or higher level compared with BT. CONCLUSION: Electric pulse stimulation is a fast, inexpensive, easy-to-use platelet activation method for autologous platelet gel therapy.

Nonthermal atmospheric plasma rapidly disinfects multidrug-resistant microbes by inducing cell surface damage.

Plasma, a unique state of matter with properties similar to those of ionized gas, is an effective biological disinfectant. However, the mechanism through which nonthermal or "cold" plasma inactivates microbes on surfaces is poorly understood, due in part to challenges associated with processing and analyzing live cells on surfaces rather than in aqueous solution. Here, we employ membrane adsorption techniques to visualize the cellular effects of plasma on representative clinical isolates of drug-resistant microbes. Through direct fluorescent imaging, we demonstrate that plasma rapidly inactivates planktonic cultures, with >5 log(10) kill in 30 s by damaging the cell surface in a time-dependent manner, resulting in a loss of membrane integrity, leakage of intracellular components (nucleic acid, protein, ATP), and ultimately focal dissolution of the cell surface with longer exposure time. This occurred with similar kinetic rates among methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. We observed no correlative evidence that plasma induced widespread genomic damage or oxidative protein modification prior to the onset of membrane damage. Consistent with the notion that plasma is superficial, plasma-mediated sterilization was dramatically reduced when microbial cells were enveloped in aqueous buffer prior to treatment. These results support the use of nonthermal plasmas for disinfecting multidrug-resistant microbes in environmental settings and substantiate ongoing clinical applications for plasma devices.

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation.

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10ns, 150kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by approximately 2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.

Ultrashort electric pulse induced changes in cellular dielectric properties.

The interaction of nanosecond duration pulsed electric fields (nsPEFs) with biological cells, and the models describing this behavior, depend critically on the electrical properties of the cells being pulsed. Here, we used time domain dielectric spectroscopy to measure the dielectric properties of Jurkat cells, a malignant human T-cell line, before and after exposure to five 10ns, 150kV/cm electrical pulses. The cytoplasm and nucleoplasm conductivities decreased dramatically following pulsing, corresponding to previously observed rises in cell suspension conductivity. This suggests that electropermeabilization occurred, resulting in ion transport from the cell's interior to the exterior. A delayed decrease in cell membrane conductivity after the nsPEFs possibly suggests long-term ion channel damage or use dependence due to repeated membrane charging and discharging. This data could be used in models describing the phenomena at work.