Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

细胞外囊泡 (EV) 是几乎所有细胞类型都会释放的纳米级脂质双层囊泡。EV 具有多种生物活性，从发育和稳态到癌症进展的作用，这刺激了 EV 作为疾病生物标志物和药物纳米载体的发展。然而，由于电动汽车尺寸较小，大多数研究都依赖于从生物流体中分离出的大量电动汽车的分离和生化分析。尽管信息丰富，但这些方法并未捕获 EV 释放、生物分布和对病理生理学的其他贡献的动态。活体和高分辨率显微镜技术的最新进展，结合创新的 EV 标记策略和报告系统，为在体内生理环境和单囊泡水平上研究 EV 提供了新工具。在这里，我们批判性地回顾了 EV 成像的最新进展和挑战，并确定了在我们寻求解开 EV 生物学和治疗应用的过程中紧迫、突出的问题。