Asymmetrical bipolar nanosecond electric pulse widths modify bipolar cancellation.

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.

Quantifying pulsed electric field-induced membrane nanoporation in single cells.

Plasma membrane disruption can trigger a host of cellular activities. One commonly observed type of disruption is pore formation. Molecular dynamic (MD) simulations of simplified lipid membrane structures predict that controllably disrupting the membrane via nano-scale poration may be possible with nanosecond pulsed electric fields (nsPEF). Until recently, researchers hoping to verify this hypothesis experimentally have been limited to measuring the relatively slow process of fluorescent markers diffusing across the membrane, which is indirect evidence of nanoporation that could be channel-mediated. Leveraging recent advances in nonlinear optical microscopy, we elucidate the role of pulse parameters in nsPEF-induced membrane permeabilization in live cells. Unlike previous techniques, it is able to directly observe loss of membrane order at the onset of the pulse. We also develop a complementary theoretical model that relates increasing membrane permeabilization to membrane pore density. Due to the significantly improved spatial and temporal resolution possible with our imaging method, we are able to directly compare our experimental and theoretical results. Their agreement provides substantial evidence that nanoporation does occur and that its development is dictated by the electric field distribution.

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP).

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.