Illuminating Extracellular Vesicles Biology with Super-Resolution Microscopy: Insights into Morphology and Composition

Super-Resolution Microscopy (SRM) is emerging as a powerful and innovative tool for imaging, characterizing, and understanding the structure of Extracellular Vesicles (EVs). By addressing the need for single-particle analysis with the high resolution required to study the composition and organization of these nanoparticles, SRM provides powerful insights into EV biology. However, its application is accompanied by significant challenges, ranging from an experimental setup to data analysis. This perspective outlines the fundamentals of SRM and its position within the broader field of EV research. We then explore their applications in evaluating (i) the morphological structure of EVs, (ii) their molecular composition, and (iii) their roles in biological systems. By offering practical guidance and an overview of critical parameters for standardization, this review aims to provide researchers with the tools and insights necessary to effectively apply SRM to EV investigation.