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These studies collectively reveal that a multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations and/or to interrogate small subsets of EVs of interest within larger EV populations in biological samples.The transfer of microRNAs (miRs) via extracellular vesicles (EVs) is a functionally relevant mechanism of intercellular communication that regulates both organ homoeostasis and disease development. Little i