No Dye Fits All: Illuminating the Challenges of Fluorescent Extracellular Vesicle Labeling.

The widespread use of fluorescent dyes for extracellular vesicle (EV) labeling has substantially improved our ability to trace EVs in vitro, ex vivo, and in vivo. However, interpretability of these experiments remains limited by methodological challenges, including incomplete dye removal, dye-associated artifacts, and intrinsic EV heterogeneity. In this study, we evaluated a range of mechanistically distinct fluorescent dyes, including lipophilic membrane- (Aco-520, DiD, FM4-64, PKH26) and protein-associated dyes (CFSE, Cy5), across EVs of both prokaryotic and eukaryotic origin. EV labeling was assessed at the single-vesicle level by nano-flow cytometry, complemented by super-resolution microscopy for visual confirmation. Our results demonstrated that EV labeling efficiency, fluorescence intensity, and background signal are not solely determined by dye chemistry but are also strongly influenced by EV biological origins. Furthermore, free dye removal and purification strategies markedly influenced nano-flow cytometry readouts, underscoring the need to utilize and optimize approaches such as ultracentrifugation and size-exclusion chromatography to effectively remove residual dye while minimizing EV loss and artifactual signals. Across all conditions, appropriate controls were critical to distinguish bona fide EV-associated fluorescence from artifactual events. Overall, our findings reinforce that no single dye is universally suitable for EV labeling and underscore the necessity of a question-driven, context-aware experimental design. We therefore include a box with key questions to support informed decision-making and improve rigor in EV labeling workflows. Addressing these guiding questions prior to experimentation, together with transparent reporting in platforms such as EV-TRACK will improve reproducibility, data interpretation, and the robustness of EV research.