Extracellular vesicles (EVs) are not only intensively studied to increase our fundamental knowledge on their functioning, but also for diagnosis, therapeutics and drug delivery purposes. To improve the current and potential applications of EVs, a fundamental understanding of their stability, structure, and function is crucial. Such studies can be conducted at the single particle level to gain biological and physical information about the vesicles and the particle to particle variability. A suitable technique to investigate EVs under near- to physiological conditions is atomic force microscopy (AFM). Operated in liquid, it provides images of the EVs while mechanical properties of the particles can be obtained as well. Here we present our app...