Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

中文翻译：

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细胞外囊泡的表型分析：荧光应用综述。

细胞外囊泡（EV）在医疗保健和诊断领域具有众多潜在应用，对其生物学功能的研究正在迅速增加。主要是因为它们的体积小和异质性，它们的分析存在重大挑战，尽管有明显的证据表明 EV 在临床诊断实践中的潜力，但分析程序指南尚未正确建立。在这里，我们概述了基于荧光原理研究电动汽车特性的主要方法。撇开分离、纯化和物理化学表征策略来回答有关电动汽车的大小、表面电荷和稳定性问题（在其他地方综述），我们重点关注可用的光学工具，这些工具能够直接分析表型和与组织相互作用的机制。简而言之，我们阐述的主题范围从最流行的方法（如纳米颗粒跟踪分析和流式细胞术）到不太常用的技术（如荧光去偏和微阵列）以及新兴领域（如快速荧光寿命成像显微镜 (FLIM)） 。我们强调，了解每种方法的优点和局限性对于选择最合适的分析工具组合至关重要。最后，讨论了医学诊断这一快速发展领域的未来方向。