Real-Time Imaging and Tuning Subcellular Structures and Membrane Transport Kinetics of Single Live Cells at Nanosecond Regime

Abstract

We developed an electric-field exposure microchannel system with 230-nanometer thin-layer gold electrodes, and interfaced it with a single living cell imaging station and a 10-nanosecond-electric-pulse (10nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport and viability of single leukemic cells (HL60) while the cells are exposed to 10nsEPs of 0–179 kV/cm, permitting the study of subcellular responses at nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10nsEPs, offering unprecedented high spatial resolution (230-nm at z-direction of E and imaging plane) for imaging intracellular molecules of single cells affected by 10nsEPs. We found that nucleic acids, membrane transport rates and viability of single cells depend on the number and electric-field-strength (E) of 10nsEPs, showing the cumulative effect of 10nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using lower E (51 kV/cm) of 10nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124 and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10nsEPs in a non-linear fashion, showing that critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity.

Microsecond and nanosecond electric pulses in cancer treatments

“…As already presented, the nanopulses should have little effect on the plasma membrane integrity. Numerous in vitro studies have confirmed this hypothesis on several cell lines: Jurkat lymphocyte cells [Ibey et al, 2010], HCT116 colon carcinoma cells [Hall et al, 2005], HL-60 leukemia cells [Xu et al, 2009], or B16F1 melanoma cells [Napotnik et al, 2010]. At higher electric fields or with an increased number of pulses, the cell and its organelles are irreversibly porated and undergo necrosis or apoptosis Ibey et al, 2010].…”

“…Because this protein is released from mitochondria it implies that the apoptosis induced by nsPEF is dependent on mitochondria, but it remains unclear if mitochondria are directly responding to the interaction with the electric field or if their involvement is a consequence of another primary event [Beebe et al, 2003a]. Different studies on leukemic cells (HL-60) have indicated that pulses as short as 10 ns also induce the condensation of intranuclear nucleic acids [Xu et al, 2009]. The effects of nsPEF have proven to be highly dependent on the electric field and pulse number but in a nonlinear fashion.…”

“…In particular, sequential pulses have cumulative effects on intracellular structures. Therefore, lower fields are needed to manipulate internal organelles without affecting the cell viability [Xu et al, 2009]. The apoptotic cell death mechanism is particularly interesting for tumor ablation.…”

Electric pulses to prepare feeder cells for sustaining and culturing of undifferentiated embryonic stem cells

“…The same number of the cells was used for control experiments. We exposed the cells to 25 sequential 10nsEPs of 150, 40 and 25 kV/cm (1-s pulse interval), provided by a 10-ns pulse generator (ARC Technology) as described previously [16]. The 10nsEPs were measured in real time using an oscilloscope (TDS 3052B, Tektronix) [16].…”

“…Recent studies show that ultrashort electric pulses (e.g., 10 ns) can penetrate the cells and induce intracellular responses while maintaining viability of cells [16,17]. The intracellular structures and viability of cells depend on the number and electric-field strength (E) of the 10-ns electric pulses (10nsEPs), showing the possibility of tuning them to prepare a wide variety of viable growth-arrested cells.…”