Uncovering Subpopulations in Extracellular Vesicles via Multiplexed Spectral PAINT Super-Resolution Microscopy.

Extracellular vesicles (EVs) are attracting growing interest in nanomedicine as nature's own nanocarriers. EV characterization remains challenging, due to complexity and heterogeneity. As such, there is a growing need for experimental techniques that can probe with molecular specificity at the single-particle level. We present a fluidic and super-resolution imaging platform that employs spectral point accumulation for imaging in nanoscale topography (sPAINT) to perform multiplexed imaging of individual EVs derived from prostate cancer (PC3) cells. By combined analysis of the solvatochromic dye Nile Red and fluorescent lectin probes, we can map glycosylation patterns and membrane polarity at the single-particle level, two rather unexplored properties of EVs. Our results reveal pronounced inter- and intraparticle heterogeneity, with polarity signatures consistent with ordered lipid domains. This multiplexed single-molecule imaging strategy enables truly multiparametric EV analysis, allowing the identification of subpopulations that remain indistinguishable in bulk or single-marker approaches.