Abstract
Extracellular vesicles (EVs), secreted from most cells, are small lipid membranes of vesicles of 30 to 1000 nm in diameter and contain nucleic acids, proteins, and intracellular organelles originating from donor cells. EVs play pivotal roles in intercellular communication, particularly in forming niches for cancer cell metastasis. However, EVs derived from donor cells exhibit significant heterogeneity, complicating the investigation of EV subtypes using ensemble averaging methods. In this context, we highlight recent studies that characterize individual EVs using advanced techniques, including single-fluorescent-particle tracking, single-metal-nanoparticle tracking, single-non-label-particle tracking, super-resolution microscopy, and atomic force microscopy. These techniques have facilitated high-throughput analyses of the properties of individual EV particles such as their sizes, compositions, and physical properties. Finally, we address the challenges that need to be resolved via single-particle (-molecule) imaging and super-resolution microscopy in future research.