guidelines suggests that EV researchers use the term small EVs (sEVs) for vesicles that are less than 200 nm in diameter. [4] sEVs contain many essential cargo biomolecules such as nucleic acids (DNA, mRNA, microRNA), proteins, and lipids. [1,3,5] sEVs have emerged as a functional mediator for communications between cells in health and disease. [1,6] sEVs derived from tumor cells contain disease-specific proteins, RNA, and double-stranded DNA (dsDNA), thereby representing the disease state and progression. [1,2,[7][8][9] Especially in tumor microenvironment, sEVs transfer their cargo from the tumor to stromal cells. [10,11] sEVs play pathophysiological roles in many other diseases, including neurodegenerative diseases, and various infections. They transmit important biomolecules that regulate many biological processes and influence the immune system. [1,[12][13][14][15] In the last decade, studies dealing with sEV biogenesis pathways and the role of sEVs in health and disease have grown exponentially. However, exploring the released sEVs and interaction of their cargo with cellular biomolecules in the distant recipient cells is significantly hampered by the lack of microscopy studies using improved imaging technology. Most of the current microscopy studies involving sEVs use conventional confocal microscopy to generate either 2D or 3D reconstruction images using Imaris. [11,[16][17][18][19][20][21][22] Although these images provide the information Small extracellular vesicles (sEVs) are 30-200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.