Gorti, V.; Serafini, C. E.; Silva Trenkle, A. D.; McCubbins, K.; LeCompte, I.; Kwong, G. A.; Robles, F. E.

T cell characterization is critical for understanding immune function, monitoring disease progression, and optimizing cell-based therapies. Current technologies to characterize T cells, such as flow cytometry, require fluorescent labeling and typically are destructive endpoint measurements. Non-destructive, label-free imaging methods have been proposed, but face limitations with throughput, specificity, and system complexity. Here we demonstrate deep-ultraviolet (UV) microscopy as a label-free, non-destructive, fast and simple imaging approach for assessing T cell viability, activation state, and subtype with high accuracy. Using static deep-UV images, we characterize T cell viability and activation state, demonstrating excellent agreement with flow cytometry measurements. We further apply dynamic deep-UV imaging to quantify intracellular activity, enabling fast and accurate subtyping of CD4 and CD8 T cells. These results corroborate recent studies on metabolic activity differences between these subtypes, but now, with deep-UV microscopy, they are enabled by a non-destructive, fast, low-cost and simple approach. Together, our results demonstrate deep-UV microscopy as a powerful tool for high-throughput immune cell characterization, with broad applications in immunology re-search, immune monitoring, and development of emerging cell-based therapies.