Comparing label-free and fluorescent indicators for sensitivity to pulsed electric field-induced cell damage

The effects of nano-second pulsed electric fields (nsPEFs) on cells are an expanding area of research. nsPEFs have been utilized in multiple areas, but research has primarily focused on medical applications where they have been explored for tumor treatment and tissue ablation. Understanding the point of onset of these unique bioeffects is vital to determining safety and efficacy for patients and to ensure proper dosimetry. One limiting factor in understanding the effects of nsPEFs on cells is the ability to characterize subtle damage induced by nsPEFs near the damage threshold. Fluorescence microscopy using a membrane exclusionary dye to indicate membrane damage is the current gold standard, but is limited to mostly qualitative measurements, and it is unclear if the onset of nsPEF-induced damage is best measured using fluorophore uptake or some other metric. Quantitative phase imaging (QPI) is a modality that has emerged as a sensitive, label-free alternative to fluorescence microscopy that can quantify metrics such as cell mass, density, water content, and other biophysical properties. QPI and fluorescence microscopy have not been directly compared to assess sensitivity to cell damage in any past work. The following proceeding details the design and construction of a dual-modality QPI and fluorescence microscopy system with an integrated electrode pair capable of delivering nsPEF pulses. The system was leveraged to conduct a sensitivity analysis to compare the onset of detectable change induced by a variable number of nsPEF pulses. The results suggest that fluorescence microscopy is more sensitive to cell damage overall and senses this damage faster than QPI. However, more data is needed with this specific system, and the generalizability of these results will require additional experiments across varying cell types and stimuli.