FLUORESCENCE IMAGING IN THE MILLISECOND TIME RANGE OF MEMBRANE ELECTROPERMEABILISATION OF SINGLE CELLS USING A RAPID ULTRA-LOW-LIGHT INTENSIFYING DETECTION SYSTEM

A fast and sensitive fluorescence image acquisition system is describe d which uses an ultra-low-light intensifying camera able to acquire di gitised fluorescence images with a time resolution of 3.33 ms/image. T wo modes of recording were employed. The synchronisation mode allowed acquisition of six successive 3.33 ms-images synchronised with an exte rnal trigger, while the memorisation mode allowed acquisition of twelv e successive 3.33 ms images starting after a 20 ms-time lag from the e xternal trigger. Interaction of ethidium bromide (EB) with the membran e of electropermeabilised living cells was studied using this imaging system. We observed enhanced fluorescence of the dye when associated w ith 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 sh own to represent the electropermeabilised area of the cell membrane. T he 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 intera ction with the membrane was achieved in a maximum 20 ms-time lag after electropermeabilisation. The membrane labelled parts were always obse rved in the cell regions facing the electrodes. They were present only when the electric field strength was higher than a threshold value wh ich was different for the two cell sides. An increase in electric fiel d intensity led to an increase in the dimensions of the labelled cell region.