Single-molecule localization microscopy imaging of extracellular vesicle DNA in recipient cells

Small extracellular vesicles (sEVs) are critical mediators of intercellular communication in both physiological and pathological contexts, including cancer, by transporting key biomolecules between cells. However, the biogenesis, packaging, and functional roles of DNA associated with sEVs (EV-DNA) remain poorly understood, largely due to the lack of efficient EV-DNA labeling dyes compatible with super-resolution imaging techniques. Here, we employed BODIPY, a green-emitting, buffer-independent blinking fluorophore, to label EV-DNA cargo and applied single-molecule localization microscopy (SMLM) for the first time under physiological conditions to achieve nanoscale imaging of EV-DNA in recipient cells. This approach overcomes conventional fluorophore limitations, enabling high-resolution dual-color imaging without quenching artifacts. We further developed a co-labeling strategy combining click chemistry (EV-DNA) and nanobody-based immunostaining (CD63-GFP⁺-sEVs), achieving precise multi-target labeling with a calculated linkage error of ~ 2 nm. Dual-color SMLM imaging revealed limited co-localization between EV-DNA and CD63-GFP⁺-sEVs, suggesting that EV-DNA may associate with distinct vesicle populations. Additionally, dual-color SMLM combined with cluster analysis indicated partial spatial proximity between EV-DNA and the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), suggesting potential but limited functional interactions. The SMLM-based imaging approach established in this study provides a powerful platform for investigating the packaging and subcellular fate of EV-DNA at nanometer resolution. Our results uncover new aspects of EV-DNA biology, including limited association with CD63⁺ vesicles and partial proximity to cGAS, suggesting alternative intracellular pathways. This versatile approach will enable detailed exploration of EV-DNA dynamics and its functional roles in health and disease.