Mammalian cell electropermeabilization as revealed by millisecond imaging of fluorescence changes of ethidium bromide in interaction with the membrane

Molecular events associated with membrane electropermeabilization remain po orly known. Video-microscopy appears as a promising approach for their inve stigation, but a fast time resolution is therefore needed. We developed a f ast and sensitive fluorescence image acquisition system which used an ultra -low-light intensifying camera working with a very short time resolution (3 00 images/s). Interaction of ethidium bromide (EB) with the membrane of ele ctropermeabilized Chinese hamster ovary (CHO) cells was studied using this imaging system. Altered parts of the single cell membrane were identified v ia the enhancement in fluorescence intensity of the dye. 3.33 ms-images of fluorescence patterns reflected the electropermeabilized parts of the cell membrane, and revealed an asymmetrical permeabilization of the cell. During millisecond-pulses, membrane labelling was always only observed on the ano de-facing cell hemisphere as soon as the electric field was higher than a t hreshold value. Nevertheless, within at most 40 ms after the end of the pul se, the electro-induced labelling of the electropermeabilized cell was diff erent. The anode-facing labelling was still present as soon as the field wa s higher than the same threshold value but when the electric field was over a second higher threshold value, the two electrode-facing cell sides were labelled, the part of the labelled anode-facing side being larger. During t he pulse, electropermeabilization of living cell membranes is an asymmetric al process. Post-pulse electropermeabilization of membrane is affected by i ts resting transmembrane electrical potential. The phenomenon is local on t he cell surface and the fraction of the membrane on which structural altera tions occurred was controlled only by the electric field strength. (C) 1998 Elsevier Science S.A. All rights reserved.