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

Abstract

Molecular events associated with membrane electropermeabilization remain poorly known. Video-microscopy appears as a promising approach for their investigation, but a fast time resolution is therefore needed. We developed a fast and sensitive fluorescence image acquisition system which used an ultra-low-light intensifying camera working with a very short time resolution (300 images/s). Interaction of ethidium bromide (EB) with the membrane of electropermeabilized Chinese hamster ovary (CHO) cells was studied using this imaging system. Altered parts of the single cell membrane were identified via 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 anode-facing cell hemisphere as soon as the electric field was higher than a threshold value. Nevertheless, within at most 40 ms after the end of the pulse, the electro-induced labelling of the electropermeabilized cell was different. The anode-facing labelling was still present as soon as the field was 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 the pulse, electropermeabilization of living cell membranes is an asymmetrical process. Post-pulse electropermeabilization of membrane is affected by its resting transmembrane electrical potential. The phenomenon is local on the cell surface and the fraction of the membrane on which structural alterations occurred was controlled only by the electric field strength.