A team of researchers from Georgia Tech and Emory University has developed a deep ultraviolet (UV) microscopy method that can quickly assess T cell viability, activation status, and subtypes without the need for fluorescent labeling or cell destruction. The study, published in BME Frontiers, provides an innovative approach to immune monitoring and the development of cell-based therapeutics.
T cells are central to the immune system, and their characterization is important for understanding immune function, tracking disease progression, and optimizing adoptive T cell therapies such as CAR-T. However, current leading methods such as flow cytometry require fluorescent labeling and expensive equipment, and typically destroy cells during measurements. This limits real-time monitoring and long-term studies of live cell cultures.
The new approach uses static deep UV images acquired at 255 nm, a wavelength that is strongly absorbed by nucleic acids, to generate high-contrast images of live T cells free of exogenous stains. By training a custom residual neural network on images from five human donors, the researchers achieved high accuracy in classifying T cells into three categories: activated, dead, and quiescent (naive or systolic). Model predictions showed excellent agreement with flow cytometry, with R² > 0.97 for both survival and activation percentage.
A more difficult challenge is subtyping CD4+ helper T cells from CD8+ cytotoxic T cells. Static morphological features alone proved insufficient. To overcome this, the team turned to dynamic deep ultraviolet imaging, acquiring a time series of 500 frames at approximately 8 Hz. They quantified intracellular activity by analyzing pixel-by-pixel temporal variations in the frequency domain using phasor analysis and power-law fitting. A second neural network fed four channels of input (UV absorption, phasor g, phasor s, and power law slope) differentiated between CD4+ and CD8+ T cells with approximately 90% accuracy.
Notably, consistent with known metabolic differences, CD4+ T cells exhibited significantly higher intracellular dynamic activity than CD8+ cells. CD4+ cells rely more on glycolysis and oxidative phosphorylation and have more cytoplasmic mitochondria. Pseudocolor images revealed that the activity differences were localized to the cytoplasm rather than the nucleus, further supporting the link to metabolic mechanisms.
The potential applications of deep ultraviolet microscopy are vast, spanning immunological research, immune monitoring, and the development of new cell-based therapies. Its simplicity, speed, and high resolution make it a valuable tool for optimizing adoptive T cell therapy, tracking disease progression, and enhancing fundamental understanding of immune function.
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Reference magazines:
Gorty, V. others. (2026). Non-destructive, high-resolution T cell characterization and subtyping using deep ultraviolet microscopy. BME frontier. DOI: 10.34133/bmef.0227. https://spj.science.org/doi/10.34133/bmef.0227
