Advanced optical methods and strategies for extracellular vesicles characterization and dynamic studies

Extracellular vesicles (EV) can serve as nanocarriers for drug delivery, but their clinical translation is hampered by challenges in characterizing their dynamic interactions with target cells. Moreover, the dynamic reaction between EV and target cells remains unclear, especially at single-particle level. Recent techniques in advanced microscopy and artificial intelligence (AI) now present an opportunity to overcome this bottleneck. Therefore, the goal of this review is to evaluate how these technologies can be integrated to provide a dynamic, and label-free understanding of EV biology. In this review, we discuss the properties of the EV and commonly used probes for EV tracking. We also review the latest advances in microscopic techniques for visualizing EV uptake. Furthermore, we explore the potential of AI driven image analysis from these complex processes. By emphasizing the importance of these techniques, this review aims to provide a clear roadmap for researchers to leverage these new tools and ultimately provide new perspectives for EV-based therapeutics.