Unveiling Heterogeneity: Innovations and Challenges in Single‐Vesicle Analysis for Clinical Translation

ABSTRACT Extracellular vesicles (EVs) are key mediators of intercellular communication, carrying diverse molecular cargo that reflects the dynamic physiological and pathological state of their source cell. While analyses of the entire vesicular population (bulk EV) have advanced our understanding of their roles in health and disease, these approaches often obscure the heterogeneity inherent in EV populations. Emerging single‐vesicle analysis technologies offer unprecedented resolution, enabling the identification of individual EV subpopulations and their distinct molecular signatures. Such approaches, combined with digital platforms, can now analyze individual molecules from single EVs, including single‐molecule features such as protein, mRNA, double‐stranded DNA and single‐stranded DNA. This perspective explores the transformative potential of single EV technologies in clinical diagnostics and therapeutic applications. We highlight key advancements including microfluidic platforms, super‐resolution microscopy and AI‐driven data analyses, that are shaping and advancing the field and its applications. With the development and advancement of clinically viable single EV technologies, we are beginning to appreciate the complexity and abundance of cell type and specific EVs. We further discussed the challenges of sensitivity, specificity, standardization and scalability hindering these technologies' broad acceptance and feasibility in clinical translation. This perspective paper originates from discussions at the Chinese Society of Extracellular Vesicles (CSEV) annual meeting, held in Guangzhou, China, on 16 November 2024. At this meeting, researchers from various fields of EV research, with a particular emphasis on single EV digital, analytical and quantitative technological platforms, discussed the opportunities and challenges of this emerging single‐EV‐focused technology. The paper aims to provide a roadmap for integrating single EV technologies into routine EV‐research and even clinical practice, paving the way for novel scientific and diagnostic tools, personalized therapies, and a deeper understanding of EV heterogeneity and EV biology.