High-Resolution Imaging of Single Extracellular Vesicles

To obtain sub‐diffraction limit resolution images of single Evs, 1 × 10⁹ Evs in 1 μl were stained with a ‘tetramix’ of anti‐CD9, (R&D Systems, USA), anti‐CD63, (Becton Dickinson, USA) and anti‐ CD81 (R&D Systems, USA), all conjugated to AlexaFluor 647 (Gomes et al., 2022 (link)) or with antibodies supplied in the EV profiler Kit (Oxford Nanoimaging, UK) including anti‐CD9‐ATTO488, anti‐CD63‐Cy3 and anti‐CD81‐AlexaFluor647. Samples were processed according to manufacturer's instructions to immobilise the stained Evs on the chips provided with the ONI EV profiler kit. Three fields of view were recorded for each sample using direct stochastic optical reconstruction microscopy (dSTORM) on a Nanoimager S Instrument (Oxford Nanoimaging, UK) using 40%, 20% and 20% power for the 488 nm, 561 nm and 640 nm laser, respectively. For localisation mapping, 2500 images were recorded per channel. To quantify the images and to detect colocalisations, we either used the EVAnalyzer software as described in the manuscript, or the CODI platform (Oxford Nanoimaging, UK) as described previously (Wolf et al., 2022 (link)).