Fluorescence imaging in the millisecond time range of membrane electropermeabilisation of single cells using a rapid ultra-low-light intensifying detection system

Abstract

A fast and sensitive fluorescence image acquisition system is described which uses an ultra-low-light intensifying camera able to acquire digitised fluorescence images with a time resolution of 3.33 ms/image. Two modes of recording were employed. The synchronisation mode allowed acquisition of six successive 3.33 ms-images synchronised with an external trigger, while the memorisation mode allowed acquisition of twelve successive 3.33 ms images starting after a 20 ms-time lag from the external trigger. Interaction of ethidium bromide (EB) with the membrane of electropermeabilised living cells was studied using this imaging system. We observed enhanced fluorescence of the dye when associated with electropermeabilised cells. Using single cells, 3.33 ms-images of the fluorescence interaction patterns of ethidium bromide showed well-defined membrane labelling. The enhanced fluorescence patterns were shown to represent the electropermeabilised area of the cell membrane. The average level of fluorescence associated with the labelled part of the cell membrane increased linearly during and immediately (less than 7 ms) after the electropermeabilisation pulse. Steady-state EB interaction with the membrane was achieved in a maximum 20 ms-time lag after electropermeabilisation. The membrane labelled parts were always observed in the cell regions facing the electrodes. They were present only when the electric field strength was higher than a threshold value which was different for the two cell sides. An increase in electric field intensity led to an increase in the dimensions of the labelled cell region.