Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and colocalization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

Immunostaining of extracellular vesicles (EVs) has become necessary for the characterization of EV subtypes, clarification of the EV biogenesis/cellular uptake pathway, drug delivery, etc. Immunostained EVs must be in suspension for further downstream analyses or uses. However, conventional EV immunostaining methods yielding EVs in suspension lack either sufficient recovery or staining specificity because of the washing steps. In this study, we have devised and tested a method to wash immunostained EVs with successive aqueous two-phase system (ATPS) separations. The ATPS is a liquid–liquid extraction procedure that ensures a gentle separation of target molecules. The ATPS has been successfully employed to separate EVs from other impurities with high yield and high purity. Immunostained EVs were washed with the ATPS and compared with other immunostaining methods to confirm the proposed method’s high EV recovery and staining accuracy. According to the result, the ATPS-based EV immunostaining method required as low as ∼1 μg without compromise of accuracy and recovery.

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