Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles with key roles in cell biology and growing importance in biomedicine. However, EVs are challenging to characterize because they are highly heterogeneous. Single EV analysis methods are valuable tools for addressing this challenge. Here, we developed single EV analysis pipeline for assessment of transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM) images. Our analysis of morphology and locali