High electric field-treated cells are permeable to molecular dye through either opening of pores in the plasma membrane or other unknown processes which can disturb the membrane in an organized way. However, direct morphological evidence is lacking and responses of intracellular organelles are not clear. We used traditional chemical fixatives and biochemical techniques to capture cell membrane and organelle changes immediately after pulsing with high voltage electric field application. Different pulse durations, nanosecond (ns) and microsecond (µs), and field magnitudes, 60 kV/cm and 1.2 kV/cm, were applied to mouse melanoma B16-F10 cells. Two different ns durations (60 and 300 ns) with an electric field of 60 kV/cm and microsecond duration (100 µs) at 1.2 kV/cm were used in this study.

Morphological changes on plasma membranes and cell organelles were analyzed with transmission electron microscopy (TEM) immediately after one to six applied pluses. TEM micrographs demonstrated morphological changes in plasma membrane and mitochondrial structure for treated cells under certain pulse conditions. Additionally, B16-F10 cells were: I) assessed post-pulse for membrane permeability and live/dead ratio using trypan blue; 2) monitored for mitochondrial membrane potential (Δψm) changes with JC-1, a voltage-sensitive mitochondrial dye; and 3) cultured for 24 hrs post-pulse to determine long-term viability. Detailed cellular responses were evaluated based on the different electric fields, pulse duration, and number of pulses.

Cell membranes appeared to be unperturbed while mitochondrial membranes were negatively affected after the defined ns pulse treatments. Increasing the number of ns pulses introduced more mitochondrial abnormalities and led to decreased cellular viability. With fewer pulse numbers (1-2 pulses), mitochondrial morphology and Δψm were similar to controls. With µs pulse duration, intracellular organelles were less disturbed than the cell membranes. Under high electric field (60 kV/cm), changes in cell membrane permeability and irregularity increased, while cell viability and mitochondrial potential decrease, both with the longer duration (300 ns vs. 60 ns) and with higher pulse numbers under the same duration. The low electric field (1.2 kV/cm) caused fewer changes to the cell membrane and intracellular organelles even though the pulse durations (100 µs vs. 300 or 60 ns) were longer.