Biochemical characterization of cell electropermeabilization using the Raman effect

The main consequence of the interaction between cells and pulsed electric fields is the destabilization of the plasma membrane leading to its permeabilization (allowing, for example, an uptake of external molecules by the biological cells). Electrochemotherapy [1,2] is one of the medical applications of this biophysical interaction. Although known for decades, the underlying mechanisms of cells electropermeabilization are still not fully understood [3]. One hypothesis explaining the membrane destabilization is the presence of chemically altered membrane phospholipids after the electrical excitation. We detail here a highly sensitive method coupling Raman Spectroscopy and CARS microscopy, to characterize the biochemical consequence of membranes electropermeabilization. An experimental setup of Raman spectroscope has been designed to probe the vibrational footprint of the membrane phospholipids. Comparing the vibrational footprint of biological samples before and after the application of pulsed electric fields can provide critical spectrum areas to characterize electropermeabilization. At UMR8203, a unique and homemade device [4] combines nanosecond electric pulses generator and wide-field CARS microscopy allowing to acquire images of living cells with a high time resolution. Based on the critical spectrum areas previously determined with Raman Spectroscopy, it will be possible to follow the evolution of the biochemical composition of the membrane during a pulsed electric field. The study is performed on two study systems: Giant Unilamellar Vesicles (GUV) of phospholipids which are a simple mimic of the cell membrane and DC-3F cells (Chinese hamster lung fibroblast cell) which are a classical cell line used to study electropermeabilization.