Fig. 4

Super-resolution characterization of single sEVs by UCNPs labelling. a Schematic illustration of scanning a doughnut excitation beam to resolve UCNPs clusters on single sEVs. b Confocal microscopic image of UCNPs clusters. c Super-resolution image of the same UCNPs clusters as those in (b). Blue and green dashed boxes mark an area containing multiple adjacent UCNPs that can be resolved in super-resolution microscopy, but not in confocal imaging. d, e Line profiles of the UCNPs clusters from the confocal image and super-resolution image. Two clusters of doublet UCNPs can be resolved with the UCNPs particles separated by 41.9 nm and 46.7 nm, respectively. Pixel dwelling time: 1 ms. Scanning step size: 10 nm. f The percentage of the number of UCNPs per sEV resolved and quantified by super-resolution imaging in a single testing sample. There are 75.47% of singlet UCNPs, 22.64% of doublet UCNPs, and 1.89% of triplet UCNPs on single sEVs. n = 100. g The averaged luminescent intensity of bright signal spots on the number of UCNPs per single sEV (n = 100), showing the three populations of single EVs carrying one, two, or three UCNPs