LAUSR

Abstract Membrane‐bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell‐surface proteins, but a generalizable technique that can quantitatively observe these vesicle–protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell‐surface proteins, either in a surface‐tethered or in a membrane‐embedded state. By employing cell‐derived vesicles (CDVs) and intercellular adhesion molecule‐1 (ICAM‐1) as a model system, we found that integrin‐driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density‐dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles.