Comprehensive Characterization of the Interaction between Pulsed Electric Fields and Live Cells by Confocal Raman Microspectroscopy

Abstract

This study reports a comprehensive analysis of the effect of 100 μs electric pulses on the biochemical composition of live cells using a label-free approach, confocal Raman microspectroscopy. We investigated different regions of interest around the nucleus of the cells and the dose-effect relationship related to different electric pulse parameters. We also extended the study to another cell type. Membrane resealing was monitored by pulsing the cells in reversible or irreversible electropermeabilization condition at different temperatures. Our results confirmed a previous publication showing that proteins and lipids were highly impacted by the delivery of electric pulses. These chemical changes were similar in different locations around the cell nucleus. By sweeping the field magnitude, the number of electric pulses, or their repetition rate, the Raman signatures of live cells appeared to be related to the electropermeabilization state, verified by Yo-Pro-1 uptake. We also demonstrated that the chemical changes in the Raman signatures were cell-dependent even if common features were noticed between the two cell types used.

Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods

“…1b). Confirming our previous studies 21,22 , several Raman vibrations were affected by the delivery of the electric pulses. The phenylalanine ring breathing vibrational mode at 1003 cm −1 and the Amide I band at 1658 cm −1 were the predominant peaks.…”

“…We may attribute the three groups (first: 0 and 500 V/cm; second: 750, 1000 and 1250 V/cm; third: 1500 V/cm), identified by statistical analysis, to three different states of the plasma membrane, meaning no permeabilization, reversible permeabilization and irreversible permeabilization. Therefore, in this manuscript, using a PLS approach, we further extend the analysis of the data reported in our previous manuscript 22 where the multivariate analysis was performed using the Principal Component Analysis (PCA) method, where a principal component is a spectrum supporting a specific variance of the data set. Whether we use PLS or PCA, 0 and 500 V/cm result in no significant change with respect to the unpulsed samples, and we can consider that no permeabilization of the membrane is detectable using CRMS.…”

“…This saline solution is commonly used for CRMS acquisitions on living cells 34 . Due to the long duration of Raman acquisitions, they were performed at 4 °C to avoid the interference of the effects of membrane resealing process 22 . For the pulse generator and pulses conditions.…”

Assessing the skin barrier via corneocyte morphometry

“…The high analytical power allows for other constituents to be profiled, for example, 2‐pyrrolidone‐5‐carboxylic acid (PCA, constituent of NMF) or topically applied agents. Even live monitoring of diffusion is possible, rendering Raman spectroscopy a valuable tool for clinical studies of various design as could be demonstrated in patients with cancer under treatment . Measuring times of <1 minutes allow for applications in larger scale studies.…”

The contribution of lipid peroxidation to membrane permeability in electropermeabilization: A molecular dynamics study

“…Nevertheless, the contribution of lipid peroxidation to the permeability of electropermeabilized cell membranes has not yet been quantitatively assessed. Indeed there are other possible mechanisms participating in the membrane permeability including changes in the conformation of membrane proteins [42][43][44][45]. To elucidate the role of lipid peroxidation in electropermeabilization such quantitative assessment needs to be carried out.…”