Endogenous Labelling of Extracellular Vesicles and Image Capture of Their Interactions With Acceptor Cells

Despite their significance in both physiological and pathological contexts, the mechanisms governing extracellular vesicle (EV) uptake and cytosolic cargo delivery remain incompletely understood. Here, we report the development of a BF 2 ‐azadipyrromethene‐based near‐infrared fluorophore for the endogenous labelling of EVs, enabling their interaction with acceptor cells to be observed using both intensity and fluorescence lifetime imaging microscopies. Through endogenous labelling, dye aggregate‐free EVs can be readily isolated and confirmed through their emission wavelengths (λ max 720 nm) and lifetime (2.7 ns). These photophysical properties permitted clear discrimination between aggregated and disaggregated forms of the fluorophore in complex biological environments. Endogenously labelled EVs containing the disaggregated fluorophore were incubated with unlabelled acceptor cells and imaged by both intensity and lifetime microscopy, confirming identical intensity and lifetime values to the free disaggregated dye. Incubation at 4°C, which slows cellular processes, enabled visualisation of initial EV–plasma membrane interactions, while at 37°C efficient transfer of the fluorophore into acceptor cells was observed, evidenced by increased emission intensity and matching characterised spectral–lifetime signatures. Our results establish endogenous labelling as an efficient method to introduce a fluorophore into the lumen of EVs, thereby providing a robust platform for real‐time visualisation of EV communication with acceptor cells.