Upconversion nanoparticles for super-resolution quantification of single small extracellular vesicles

Although small EVs (sEVs) have been used widely as biomarkers in disease diagnosis, their heterogeneity at single EV level has rarely been revealed. This is because high-resolution characterization of sEV presents a major challenge, as their sizes are below the optical diffraction limit. Here, we report that upconversion nanoparticles (UCNPs) can be used for super-resolution profiling the molecular heterogeneity of sEVs. We show that Er 3+ -doped UCNPs has better brightness and Tm 3+ -doped UCNPs resulting in better resolution beyond diffraction limit. Through an orthogonal experimental design, the specific targeting of UCNPs to the tumour epitope on single EV has been cross validated, resulting in the Pearson’s R-value of 0.83 for large EVs and ~ 65% co-localization double-positive spots for sEVs. Furthermore, super-resolution nanoscopy can distinguish adjacent UCNPs on single sEV with a resolution of as high as 41.9 nm. When decreasing the size of UCNPs from 40 to 27 nm and 18 nm, we observed that the maximum UCNPs number on single sEV increased from 3 to 9 and 21, respectively. This work suggests the great potentials of UCNPs approach “digitally” quantify the surface antigens on single EVs, therefore providing a solution to monitor the EV heterogeneity changes along with the tumour progression progress.