Spatiotemporal fate of nanoplastics: From membrane encounter to perinuclear confinement revealed by STED microscopy.

Nanoplastics (NPs) are ubiquitous environmental contaminants, yet their subcellular fate from membrane encounter to destination remains poorly understood due to limitations in imaging resolution. Here, we address this gap by using stimulated emission depletion (STED) super-resolution microscopy to visualize the complete intracellular journey of individual NPs at nanoscale precision. A photostable AIEgen-core NP enabled resolution of individual NPs and real-time tracking of their nanoscale spatial relationships with subcellular structures. Using a methodologically elevated NP concentration, live-cell STED revealed that internalization was an active, migration-driven process involving filopodia-initiated probing, partial engulfment followed by particle release, and successful perpendicular uptake, challenging assumptions that membrane contact necessarily leads to bioaccumulation. Intracellularly, NPs moved rapidly within lysosomes toward the perinuclear region (peak speeds ∼0.15 μm/s), undergoing fusion dynamics that modulated mobility. Mitochondria exhibited transient "kiss-and-run" NP contact, which could be associated with stress-induced donut-shaped morphology and oxidative stress elevation. Quantitative 3D projection by STED revealed that conventional 2D imaging underestimated intracellular NP burden by up to 100% under low-count conditions. High-resolution STED differentiated true lysosomal encapsulation from mere proximity, revealing a lysosome-independent pathway for nuclear-proximal NP enrichment. These findings highlight STED microscopy's potential in advancing NP risk assessment and establish a mechanistic foundation for understanding NP-induced toxicity at the subcellular level.