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Exosomes are cell-secreted nanometer-sized vesicles(30-100 nm)that have been reported to play an important role in intercellular communication.These complex structures consist of a lipid bilayer that contains transmembrane proteins and encloses soluble hydrophilic components derived from the cytosol of the donor cell.Because exosomes are considered potential diagnostic markers,therapeutic agents,and drug delivery systems,research on exosomes is of great importance in disease diagnosis and treatment.As different cell type and even different activation status of the donor cell secret exosomes of different molecular compositions,developing advanced techniques that can analyze exosomes at the single-particle level is essential to reveal the intrinsic heterogeneity of exosome populations and to illustrate the underlying molecular mechanisms of intercellular communication.Nanoparticle tracking analysis(NTA)and resistive pulse sensing(RPS)are the techniques currently available for the detection of single exosomes in suspension.However,both techniques can only detect exosomes larger than 70~90 nm in size and the resolution is very limited.Our laboratory has developed high-sensitivity flow cytometry(HSFCM)that achieves real-time light scattering detection of single silica and gold nanoparticles as small as 24 nm and 7 nm in diameter,respectively.Here,we will report how the HSFCM can be applied to the sensitive analysis of single exosomes.We extracted exosomes from human and mouse plasma using ultracentrifugation.Employing fluorescent nanoparticles of known concentration as the internal standard,we measured the concentration of exosomes via single particle enumeration at a throughput up to 10,000 particles per minute.Employing silica nanoparticles with comparable refractive index to exosomes as the sizing reference,rapid and highresolution size distribution analysis of exosomes was attained.Meanwhile,biochemical properties of single exosomes can be measured through concurrent fluorescence detection.HSFCM offers quantitative multiparameter analysis of exsosomes,and broad applications in exosome research can be envisioned.