Optical Manipulation-Assisted Three-Dimensional Single-Particle Tracking Reveals Extracellular Vesicle–Cell Real-Time Interactions In Vitro

Abstract Extracellular vesicles (EVs) play essential roles in intercellular communication and are promising nanocarriers for drug delivery. Directly resolving the three-dimensional (3D) dynamics of EVs during their interactions with target cells can provide critical insights and help guide the rational design of EV-based therapeutics. However, accurate characterization of these processes requires approaches that enable rapid manipulation, real-time monitoring, and high-sensitivity 3D imaging at the single-particle level. The ability to manipulate and measure biomolecular interactions renders label-free biosensors a critical focal point in the field of biosensing. Here, we present an optical manipulation and imaging platform based on an all-dielectric one-dimensional photonic crystal (1DPC) biosensor for label-free 3D tracking of single EV–cell interactions. Using optical tweezers, we delivered individual EVs to the vicinity of the target cell membrane. The subsequent 3D trajectories of the unlabeled EVs are recorded with a double-helix point spread function (DH-PSF) microscope. By analyzing high-resolution trajectories, we quantified EV–cell interaction dynamics at the single-vesicle level and further discriminated between two distinct internalization modes: endocytosis and membrane fusion. This integrated platform enables controlled single-EV delivery, real-time 3D tracking, and quantitative assessment of EV–cell uptake patterns in vitro. Our approach provides a versatile tool for studying EV-mediated communication and for optimizing EV-based drug delivery and therapeutic strategies.