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 mum 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

Summary form only given. Microsecond-duration pulsed electric fields (PEFs) above a certain voltage cause electroporation, or increased permeability of the cell membrane due to pore formation, while submicrosecond pulses induce intracellular effects. Models developed to describe and interpret these effects often depend on the electrical properties of the cells, which are altered by the PEF. We determined the complex permittivity of a cell suspension using time domain dielectric spectroscopy (TDDS). We used a two-shell model of the cell to calculate the conductivity and permittivity of the cell membrane, cytoplasm, nuclear envelope, and nucleoplasm from the complex permittivity. For long pulses (50 /spl mu/s), we found that cell membrane poration occurred within 10 s of the pulse, whereas poration was delayed by minutes for 10 ns pulses. These results indicate that membrane opening is the primary result for long pulses and a secondary result for ultrashort pulses, in agreement with other observations. Membrane recovery time is similar for both pulse durations. Our initial studies have focused on temporal changes in the cell membrane. TDDS will allow us to explore electrical pulse effects on the cell nucleus.

Despite a basic knowledge of cells' biochemical processes, their electrical properties, particularly the changes in membrane properties upon the application of pulsed electric fields (PEF's), have not yet been fully characterized. Microsecond pulses above a certain threshold cause electroporation of the cell membrane while nanosecond pulses of higher voltage additionally porate the inner organelles. We used Time Domain Dielectric Spectroscopy to measure the conductivity of HL-60 (human leukemia) cell suspensions as a function of time after 10 ns, 78.5 kV/cm pulses and 50 mus, 1.1 kV/cm pulses, which have the same energy. The conductivity increased immediately after the 50 mus pulse, indicating that ion channels in the HL-60 membranes initially opened. However, the conductivity decreased immediately after the ultrashort pulse, indicating that ion channels initially closed. The conductivity decreases significantly approximately 40 minutes after both pulses. This suggests that not only do the pores or channels opened close, but pores or channels open in the membrane prior to the pulse may close as well. These measurements were an intermediate step in determining the electrical properties of HL-60 cells using a two-shell model. Once determined, these electrical parameters will be used in electroporation models developed at Old Dominion University.