Fig. 5
The heterogeneity of sEVs and size limitation of UCNPs. a the theoretical number of UCNPs that can be labelled on the surface of an EV limited by the size of UCNPs. b Cryo-EM images of heterogenous sEVs isolated from HT29 cells. i-vii, single vesicles with heterogeneous size and morphology; viii-xi, double vesicles; xii-xiii, vesicles with electron-dense cargo in lumen; xiv-xv, vesicles with broken membrane (point with black arrow); xvi, double-membrane vesicles. Scale bar: 100 nm. c Positive Y axis: The measured intensity profiles along the increasing number of UCNPs per single EVs. Error bars: ± s.d. Insets: representative single diffraction-limited spots with the intensity correlated to the number of UCNPs per spot. Negative Y axis: Statistical distribution of the measured intensity profiles over the N27nm = 241 and N18nm = 144 counted spots, respectively. d Comparison between the confocal image and super-resolution image of the same area using 27 nm UCNPs. Pink box: zoom in view of the dashed pink box. Pixel dwelling time: 4 ms. Scanning step size: 10 nm. e Cross-sectional profiles of two adjacent UCNPs correspondence to the boxed region in d. f Comparison of confocal imaging and super-resolution imaging of the same area using 21 nm UCNPs. Green box: zoom in view of the dashed green box. Pixel dwelling time: 5 ms. Scanning step size: 10 nm. g Cross-sectional profiles of two adjacent UCNPs correspondence to the boxed region in f