Abstract

Pulsed electric fields (PEFs) are widely recognized as a non-thermal, selective physical therapy with wide clinical application in tumor ablation. The pulse width determines how electrical energy is distributed across plasma membrane to intracellular organelles. However, under an engineering-defined equal-dose condition (N·E2·tp), which serves as a practical control parameter rather than a measure of true cellular energy absorption, systematic and comparable experimental characterization of cellular and subcellular responses across pulse widths from the microsecond to nanosecond range remains limited. In this study, PEFs with pulse widths ranging from 100 μs to 50 ns were applied under equal-dose constraints, and cellular responses were evaluated using transmission electron microscopy (TEM), multi-organelle fluorescence imaging, and flow cytometry. The results indicate that pulse-width-dependent effects were observed under a fixed pulse-number, dose-equalized framework in which electric field strength varied across conditions. Structural and functional changes were observed in multiple organelles, including the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. Notably, nanosecond pulses were more effective in inducing mitochondrial membrane potential loss and increasing the proportion of apoptotic or non-viable cells. These findings demonstrate that, under equal-dose conditions, pulse width is a key temporal parameter governing PEF-induced biological effects, indicating that identical dose constraints do not necessarily result in equivalent biological responses. This work provides experimental foundation for parameter selection and optimization in PEF-based biomedical applications.