Fluorescence imaging of hydrogel-immobilised microalgae combined with simulations as a novel platform to study...

Cell electropermeabilisation in pulsed electric field (PEF) treatments depends on complex interactions among chamber geometry, treatment parameters, biological and environmental conditions. Traditional PEF optimisation methods, such as numerical simulations or flow cytometry (FCM), typically address only isolated aspects of these interactions. Here, we present an experimental-computational platform that combines fluorescence imaging of hydrogel-immobilised Chlamydomonas reinhardtii with electric field simulations. Thereby, local treatment intensity in complex chamber geometries can be assessed, and experimental workload is reduced by probing multiple field strengths in a single treatment. Cells embedded in a transparent hydrogel matrix were exposed to PEF using a custom grid electrode that creates spatial field gradients. After staining with Sytox Green, a marker of membrane damage, fluorescence microscopy images of the hydrogel were overlaid with simulated electric field maps. Quantitative image analysis enabled an estimation of the electropermeabilisation threshold within the semi-solid matrix and could be validated against conventional FCM in cell suspensions. During long-pulse (35 μs) exposures, some fluorescence patterns not captured by simulations highlighted the need for further investigation. Nevertheless, the platform provides a consistent, image-based method for evaluating local PEF treatment intensity and inhomogeneity, adaptable to diverse PEF chamber designs and biological systems.