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Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles

Quantitative measurements of water content within a single cell are notoriously difficult. In this work, we introduce a single-shot optical method for tracking the intracellular water content, by mass and volume, of a single cell at video rate. We utilize quantitative phase imaging and a priori knowledge of a spherical cellular geometry, leveraging a two-component mixture model to compute the intracellular water content. We apply this technique to study CHO-K1 cells responding to a pulsed electric field, which induces membrane permeabilization and rapid water influx or efflux depending upon the osmotic environment. The effects of mercury and gadolinium on water uptake in Jurkat cells following electropermeabilization are also examined.

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns–10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15–20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30–40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

The purpose of the present study is to investigate the inactivation of bioaerosols containing Bovine Coronavirus, BCov, under repetitively pulsed radio frequency (RF) electromagnetic exposure.These experiments were performed in a waveguide containing a flowing aerosol stream and were limited to a single RF waveform: ∼2 μs square envelope, 5.6 GHz, 4.8 kHz repetition rate. Aerosol streams were exposed to RF electric field amplitudes in the range of 41.9 +/- 6.2 kV/m. Under laminar flow conditions, 75% of the total collected aerosol stream spends 0.85 seconds or less in the RF exposure region.Application of the RF waveform changes mean survival rate of the aerosolized BCov by -0.58 decades (roughly a 74% reduction) and impacted the variance and standard deviation of the experimental results, with the RF exposure data showing an 800% increase in variance and 196% increase in standard deviation over the control results. Experimental results were compared to those from an analytic electromagnetic-heating inactivation model.The comparison indicated the feasibility that the observed reduction in BCov survival rate might be due to a combination of thermal effects and non-thermal electric field effects.Developing better insight into the mechanisms of inactivation is important for understanding the potential limits of efficacy for this method. Additionally, these results contribute an important baseline for the impact of electromagnetic fields on aerosolized pathogens.

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs.

The reversal of the electric field direction inhibits various biological effects of nanosecond electric pulses (nsEP). This feature, known as "bipolar cancellation," enables interference targeting of nsEP bioeffects remotely from stimulating electrodes, for prospective applications such as precise cancer ablation and non-invasive deep brain stimulation. This study was undertaken to achieve the maximum cancellation of electroporation, by quantifying the impact of the pulse shape, duration, number, and repetition rate across a broad range of electric field strengths. Monolayers of endothelial cells (BPAE) were electroporated in a non-uniform electric field. Cell membrane permeabilization was quantified by YO-PRO-1 (YP) dye uptake and correlated to local electric field strength. For most conditions tested, adding an opposite polarity phase reduced YP uptake by 50-80 %. The strongest cancellation, which reduced YP uptake by 95-97 %, was accomplished by adding a 50 % second phase to 600-ns pulses delivered at a high repetition rate of 833 kHz. Strobe photography of nanosecond kinetics of membrane potential in single CHO cells revealed the temporal summation of polarization by individual unipolar nsEP applied at sub-MHz rate, leading to enhanced electroporation. In contrast, there was no summation for bipolar pulses, and increasing their repetition rate suppressed electroporation. These new findings are discussed in the context of bipolar cancellation mechanisms and remote focusing applications.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

A substantial body of literature exists to study the dynamics of single cells exposed to short duration (

Exposures to low intensity radiofrequency electromagnetic fields (RF-EMFs, 100 kHz to 6 GHz) have been associated with both positive and negative effects on cognitive behavior. To elucidate the mechanism of RF-EMF interaction, a few studies have examined its impact on neuronal activity and synaptic plasticity. However, there is still a need for additional basic research that verify the reported effects and further our understanding of the underlying mechanisms of RF-EMFs on the neuronal system. The present study investigated changes in neuronal activity and synaptic transmission following a 60-min exposure to 3.0 GHz RF-EMF at a low dose (specific absorption rate (SAR) < 1 W/kg). We showed that RF-EMF exposure decreased the amplitude of action potential (AP), depolarized neuronal resting membrane potential (MP), and increased neuronal excitability and synaptic transmission in cultured primary hippocampal neurons (PHNs). The results show that RF-EMF exposure can alter neuronal activity and highlight that more investigations should be performed to fully explore the RF-EMF effects and mechanisms.

Exposures to radiofrequency electromagnetic fields (RF-EMFs, 100 kHz to 6 GHz) have been associated with both positive and negative effects on cognitive behavior. To elucidate the mechanism of RF-EMF interaction, a few studies have examined its impact on neuronal activity and synaptic plasticity. However, there is still a need for additional basic research that further our understanding of the underlying mechanisms of RF-EMFs on the neuronal system. The present study investigated changes in neuronal activity and synaptic transmission following a 60-min exposure to 3.0 GHz RF-EMF at a low dose (specific absorption rate (SAR) 1 W/kg). We showed that RF-EMF exposure decreased the amplitude of action potential (AP), depolarized neuronal resting membrane potential (MP), and increased neuronal excitability and synaptic transmission in cultured primary hippocampal neurons (PHNs). The results show that RF-EMF exposure can alter neuronal activity and highlight that more investigations should be performed to fully explore the RF-EMF effects and mechanisms.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ∼25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.

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.

Cell mechanical properties may undergo changes as a response to external stimuli or altered environment. Being able to map the mechanical properties over an entire cell in a contact-free and label-free manner is crucial to avoid affecting the measurements. Brillouin imaging is a non-invasive all-optical modality that can provide a distribution map of high-frequency elastic modulus and viscosity. In this report we first provide an overview of prior Brillouin cell imaging studies. We then demonstrate viscoelastic 2D and 3D cell imaging capabilities of a molecular filter-based Brillouin imaging system, with a first-time imaging application of multi-excitation (ME) Brillouin elastography.

Introduction to SPIE Photonics West BiOS conference 11640: Optical Interactions with Tissue and Cells XXXII

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC

Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, nsPEF exposure can activate second messenger cascades resulting in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 immortalized mouse myoblast cells have been demonstrated to be a useful in vitro model, by differentiating these progenitors into terminally transformed myotubes. We are not only able to further investigate the fundamental subcellular mechanisms activated by pulsed electric fields, but monitor muscle contraction as a functional output. From our previous efforts, we quantified calcium-green uptake as a measurement of cellular calcium uptake across a sweep of applied pulsed electric field voltages. To extend on these findings, we evaluated calcium dynamics in the intracellular space of myotubes. Given that sarcoplasmic reticulum efflux is required for muscle contraction, we tested the physiological role of the ryanodine receptor during pulsed electric field exposure on myotubes. By blocking the Ryanodine receptor with a competitive antagonist, we reduced nsPEF -induced calcium dynamics activation by 58.36% in media with calcium. Our results are the first to demonstrate that the Ryanodine receptor complex is a subcellular candidate responsible for generating calcium responses upon nsPEF exposure in myotubes.

Nanosecond pulsed electric fields (nsPEF) are high voltage (1-15 kV/cm) nanosecond energy waveforms that can impact cellular activity. On a physical level, a nsPEF generates transient membrane perturbations in the form of nanopores to allow cation influx resulting in localized membrane depolarization. On a physiological level, a nsPEF exposure can activate receptors and channels on the membrane as well as second messenger cascades, both of which results in subcellular modulation that lasts beyond the nsPEF duration. An ongoing challenge is to characterize the extent/sequence of physiological events induced by nsPEF exposure, and potential to interplay with physical effects induced by the pulse. In our laboratory, C2C12 mouse myoblast cells have been demonstrated to be a useful in vitro model, as it is feasible to differentiate these immortalized progenitors into terminally transformed myotubes. From previous efforts, we quantified YO-PRO -1 (YO-PRO-1) uptake as a measurement of membrane perturbation, and concluded that membrane damage is proportional to applied pulsed electric field voltage. To expand upon these findings, we evaluated to what extent YOPRO-1 uptake at the membrane is physical or physiological in nature. Interestingly, the P2X7 receptor complex has been extensively studied utilizing YO-PRO-1 uptake as marker of apoptotic activity. For this reason, we tested the role of P2X7 receptor complex activation to mediate YO-PRO-1 uptake during pulsed electric field exposure. By blocking the P2X7 receptor, we reduced nsPEF-induced YO-PRO-1 uptake by 31.57%. Our results demonstrate that the P2X7 receptor complex is a subcellular candidate responsible for YO-PRO-1 uptake upon nsPEF exposure in myotubes.

Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique confocal microscope system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 10-50 kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.

Cell suspensions of Escherichia coli and Lactobacillus acidophilus were exposed to 600-ns pulsed electric fields (nsPEFs) at varying amplitudes (Low-13.5, Mid-18.5 or High-23.5 kV cm−1) and pulse numbers (0 (sham), 1, 5, 10, 100 or 1000) at a 1 hertz (Hz) repetition rate. The induced temperature rise generated at these exposure parameters, hereafter termed thermal gradient, was measured and applied independently to cell suspensions in order to differentiate inactivation triggered by electric field (E-field) from heating. Treated cell suspensions were plated and cellular inactivation was quantified by colony counts after a 24-hour (h) incubation period. Additionally, cells from both exposure conditions were incubated with various antibiotic-soaked discs to determine if nsPEF exposure would induce changes in antibiotic susceptibility. Results indicate that, for both species, the total delivered energy (amplitude, pulse number and pulse duration) determined the magnitude of cell inactivation. Specifically, for 18.5 and 23.5 kV cm−1 exposures, L. acidophilus was more sensitive to the inactivation effects of nsPEF than E. coli, however, for the 13.5 kV cm−1 exposures E. coli was more sensitive, suggesting that L. acidophilus may need to meet an E-field threshold before significant inactivation can occur. Results also indicate that antibiotic susceptibility was enhanced by multiple nsPEF exposures, as observed by increased zones of growth inhibition. Moreover, for both species, a temperature increase of ≤ 20 °C (89% of exposures) was not sufficient to significantly alter cell inactivation, whereas none of the thermal equivalent exposures were sufficient to change antibiotic susceptibility categories.

Infrared laser (IRL) exposure can induce a rapid temperature change (fast thermal gradient or FTG) that is able to stimulate or inhibit neurons and, thereby, modify neurological functions. Despite extensive research into this effect, the fundamental mechanism(s) underlying how FTG causes neurological stimulation or inhibition remains unclear. While it is hypothesized that IRL-induced FTG acts directly on the neuronal plasma membrane (PM), it is uncertain if the neurological effects observed in previous studies are mostly derived from presynaptic effects (i.e., modifications in action potential (AP) firing) or also from postsynaptic effects (i.e., alteration of the synaptic responses of the excitatory and inhibitory neuronal receptors). In the present study, we present an analysis of FTG-mediated changes in neuronal PM, AP firing rate, and miniature postsynaptic excitatory and inhibitory currents (mEPSCs and mIPSCs). Our results suggest FTG induces changes in both presynaptic and postsynaptic neurophysiological mechanisms. Specifically, we found that, after IRL pulse (IRLP)-induced FTG exposure, the amplitudes of APs are reduced, but the rate of APs are increased. In contrast, the quantities of both mEPSCs and mIPSCs are reduced, but the peak-to-peak frequency and peak amplitudes are increased. The results outlined in this study demonstrate the impact of FTG on neurons and neuronal network. This information is critical for understanding the complexity of the effects of FTG on neurological functions and for demonstrating how post-synaptic mechanisms might play a crucial role in neurological excitation or inhibition seen following IRL pulse exposure.

Compressed ultrafast photography (CUP) is a new imaging technique which incorporates the high-speed imaging capabilities of a streak camera with principles from compressed sensing to allow for single-shot, ultrafast imaging of optical phenomena at up to 100 billion frames per second1. While this technique has primarily been utilized to observe physical phenomena, it has broad ranging application to biological imaging including fluorescence lifetime microscopy, and single-shot hyperspectral imaging2. Additionally, similar optical systems can be used to for streak camera microscopy (SCM), enabling the resolution of sub microsecond changes in cellular membrane potential in response to electrical stimulation3. Here, we present several applications for CUP based imaging and spectroscopy as a novel tool to enable more rapid biological imaging and sensing.

Cell-circuit models have suggested that nanosecond pulsed electric fields (nsPEFs) can disrupt intracellular membranes including endoplasmic reticulum (ER), mitochondria, and/or nucleus thereby inducing intrinsic apoptotic pathways. Therefore, we hypothesized that the unfolded protein response (UPR) would be activated, due to the fluctuations of ionic concentrations, upon poration of the ER membrane. Quantitative real-time polymerase chain reaction was utilized to measure changes in messenger RNA (mRNA) expression of specific ER stress genes in adult human dermal fibroblast (HDFa) cells treated with tunicamycin (TM) (known ER stress inducer) and cells exposed to nsPEFs (100, 10-ns pulses at 150 kV/cm delivered at a repetition rate of 1 Hz). For HDFa cells, results showed time-dependent UPR activation to TM; however, when HDFa cells were exposed to nsPEFs, no significant changes in mRNA expression of ER stress genes, and/or caspase gene were observed. These results indicate that although cell death can be observed under these exposure parameters, it is most likely not initiated through activation of the UPR. Bioelectromagnetics. 2018;39:491-499, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid.

High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol4,5-bisphosphate (PIP2) depletion in non-excitable cells identical to agonist-induced activation of the Gq11 coupled receptors. PIP2 is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca2+ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP2 depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1–5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM1). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP2 depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP2 reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues.

A bipolar (BP) nanosecond electric pulse (nsEP) exposure generates reduced calcium influx compared to a unipolar (UP) nsEP. This attenuated physiological response from a BP nsEP exposure is termed “bipolar cancellation” (BPC). The predominant BP nsEP parameters that induce BPC consist of a positive polarity (↑) front pulse followed by the delivery of a negative polarity (↓) back pulse of equal voltage and width; thereby the duration is twice a UP nsEP exposure. We tested these BPC parameters, and discovered that a BP nsEP with symmetrical pulse widths is not required to generate BPC. For example, our data revealed the physiological response initiated by a ↑900 nsEP exposure can be cancelled by a second pulse that is a third of its duration. However, we observed a complete loss of BPC from a ↑300 nsEP followed by a ↓900 nsEP exposure. Spatiotemporal analysis revealed these asymmetrical BP nsEP exposures generate distinct local YO-PRO®-1 uptake patterns across the plasma membrane. From these findings, we generated a conceptual model that suggests BPC is a phenomenon balanced by localized charging and discharging events across the membrane.

Previously, it was demonstrated that nanometer-sized pores (nanopores) are formed in outer cellular membranes after exposure to nanosecond electric pulses (nsEPs). We reported that plasma membrane nanoporation affects phospholipids of the cell membrane, culminating in cascading phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2) intracellular signaling. In the current study, we show that nsEPs initiated electric field (EF) dose-dependent PIP2 hydrolysis and/or depletion from the plasma membrane. This process was confirmed using fluorescent optical probes of PIP2 hydrolysis: PLCδ-PH-EGFP and GFP-C1-PKCγ-C1a. The 50% maximum response occurs with a single 600ns pulse achieving an effective dose (ED50) of EF~8kV/cm within our model cell system. At 16.2kV/cm, the ED50 for the pulse width was 484ns. Reduction of the pulse width or EF amplitude gradually reduced the observed effect, but twenty 60ns 16.2kV/cm pulses produced an effect similar to a single 600ns pulse of the same amplitude. Propidium iodide (PI) uptake after the nsEP exposure confirmed a strong relationship between EF-induced plasma membrane impact and PIP2 depletion. These results have expanded our current knowledge of nsEPs dependent cell physiological effects, and serve as a basis for model development of new exposure standards, providing novel tools for drug independent stimulation and approaches to differential modulation of key cellular functions.

We investigate the effect of nanosecond pulsed electric fields on optical breakdown thresholds for picosecond optical pulses (6ps pulse duration at wavelength of 1064nm) in aqueous solutions. Optical breakdown thresholds are determined both with and without the application of the nanosecond pulsed electric fields in water, phosphate-buffered saline solution, and a typical buffer solution used for live cell imaging. As optical breakdown at this time-scale mostly on a combination of multi-photon ionization as well as free electrons in the focal volume to achieve an avalanche or cascade ionization phenomena, we hypothesize that the application of a strong external electric field will have a significant impact on the breakdown probability at the lower-end of pulse energy levels, effectively modifying the pulse-energy breakdown threshold of the materials. Single optical pulses were triggered at specific times with respect to the leadingedge of the applied electric field to determine if there were any time-dependent effects on breakdown thresholds. Results of the experiments are examined and the physical phenomena as well as the concept “electrostatic purification” are discussed.

Nanosecond pulsed electric field (nsPEF) exposure to cells causes a myriad of bioeffects with great potential to translate into beneficial technology. However, a general lack of fundamental knowledge of how the field is interacting with the cell limits the advancement of predictive models and maximal exploitation. Despite 30 years of research, this same dearth of mechanistic understanding remains for longer pulse exposures. Fundamental to determining what is occurring as these strong electric fields are applied to cells is measuring the induced change in membrane potential on the time scale of the exposure. Such measurements are critical to validating commonly used electric circuit-based continuum models for electroporation, but have remained elusive due to limits in signal-to-noise and fluorescent reporters. In a previous publication, we described a high-speed fluorescent imaging modality that combined a streak camera and a high power laser source termed a high speed streak camera microscope (SCM) to resolve membrane charging during a single nsPEF. In this paper, we use the SCM to quantify changes in membrane potential in CHO-K1 cells exposed to unipolar and bipolar 600ns PEF within the context of the recently discovered “bipolar cancellation” phenomenon. Immediately after a unipolar pulse exposure, we see a prolonged “depolarization” of the cell that is roughly 50-100mV in amplitude. Such a prolonged depolarization is not seen in bipolar exposures nor is it predicted by membrane charging models. We postulate that this lasting membrane depolarization, seen only in unipolar pulse exposure, is either the cause of later uptake of impermeable ions or signifies the acute (during the pulse) breakdown of the plasma membrane (nanoporation). The lack of lasting depolarization in bipolar pulse exposures may be fundamental to “bipolar cancellation” and explain why uptake of ions is substantially reduced as compared to unipolar pulse exposures.

The combined effect of short (picosecond) optical and (nanosecond) electrical pulses on dielectric breakdown is investigated both theoretically and experimentally. It was demonstrated that nanosecond electrical pulses (nsEPs), being applied simultaneously with picosecond optical pulses, reduce the threshold for optical breakdown. Experimental results are discussed with respect to an extended model for opto-electrical-induced breakdown. The newly unveiled effect is expected to play a significant role in spatially confined electroporation and further advances in laser-ablation-based processes while also allowing for measurements of ambipolar diffusion constants.