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

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.