Project Summary To discover the fundamental mechanisms that govern how cells mature and differentiate, researchers need to monitor a cell's gene expression profile and measure changes over time without destroying or disrupting its growth. No current tool can meet this technological challenge with a throughput high enough to enable systems biology analyses. This project proposes to establish the feasibility of using localized electroporation for temporal sampling in individual living cells. Specifically, the nanofountain probe electroporation system will be used to identify localized molecular-transport mechanisms, employed in cell sampling, by monitoring the loss of fluorescence intensity in a tdTomato-expressing cell line. These experimental data will be used to validate model parameters in a multi-physics framework based on pore-evolution models. Single-cell RNA sequencing will be used to investigate any off-target effects due to repeated sampling in localized electroporated, bulk electroporated, and unelectroporated (control) cells. The ability to measure gene expression over time in single cells as they are maturing would be a transformative advance for cell biology, bioengineering, and biopharmaceutics.