Real-time imaging of the membrane charging of mamalian cells exposed to nanosecond pulsed electric fields

For real-time imaging of the transmembrane voltage of Jurkat cells, exposed to nanosecond pulsed electric fields, the cells were stained with a voltage sensitive membrane dye (VSD) and illuminated with a 4.8 ns long dye-laser pulse at various time during the electric field pulse. The stained cells were located in a 100 pin stainless steel electrode arrangement mounted at the stage of an inverted microscope. Due to the weak fluorescence response from the membrane an intensified CCD camera was used for image acquisition. The camera was operated in an open-shutter mode. ANNINE-6, a recently developed ultra-fast VSD, and Di-8-ANEPPS were used for transient membrane voltage monitoring. Best results could be achieved with the ANNINE-6 dye. First results indicate a clear response of the VSDs attached to the membrane in case of exposing the cells to a 60 ns long electric field pulse.