A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells

Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multi-vesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for always-on dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.

Automated summary

This study introduces a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of extracellular vesicles (EVs) secreted from living cells. The probe allows visualization of acidic EVs in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, minimizing false positive signals. Additionally, the joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways, providing mechanistic insights into EV transport.