DIRECT OBSERVATION IN THE MILLISECOND TIME RANGE OF FLUORESCENT MOLECULE ASYMMETRICAL INTERACTION WITH THE ELECTROPERMEABILIZED CELL-MEMBRANE

Interaction of two stains (propidium iodide and ethidium bromide) with electropermeabilized living Chinese hamster ovary cells is observed u sing an ultrafast fluorescence image acquisition system. The computing process is linked to an ultra-low-light intensifying camera working w ith a very short time resolution (3.33 ms per image). Altered parts of the cell membrane were identified via the enhancement in fluorescence intensity of the dyes. They reflect the electropermeabilized part of the membrane in which free Row of dye occurred. Images of the fluoresc ence interaction patterns of the two dyes, in a maximum 20-ms time lag after pulsation, reveal asymmetrical permeabilization of the cell mem brane. For electric field intensities higher than a first threshold va lue, permeabilization is always observed on the anode-facing side of t he cell. For electric field intensities over a second higher threshold value, the two electrode-facing hemispheres of the cell are permeabil ized, the hemisphere facing the anode being most permeable. These data support the conclusion that electropermeabilization of living cell me mbrane is affected by its resting potential. The asymmetrical pattern of the dye interaction is not dependent on the nature or concentration of the dye, the ionic strength of the pulsing buffer, or the duration of the pulse, The field intensity determines the fraction of the memb rane in which molecular alterations can occur. The extent of alteratio n in this localized region is determined by the duration of the pulse when a single pulse in the millisecond time range is applied.