Polarization Holographic 4D Phenotyping Reveals Nanoplastic Induced Dysfunction in Zooplankton

ABSTRACT Assessing contaminant‐induced dysfunction in complex living systems is critical, but remains technically challenging. Current methods rely predominantly on destructive endpoint readouts or low‐dimensional measurements, which often fail to detect early sublethal changes in freely behaving organisms. Here, we develop Polarization Holographic Imaging Microscopy (PHIM), a multiscale phenotyping framework that integrates polarization‐sensitive holography with computational volumetric reconstruction for label‐free analysis of living zooplankton in natural seawater. Without mechanical scanning, PHIM resolves four‐dimensional (4D) locomotor dynamics and endogenous structural anisotropy. Using marine rotifers as a model zooplankton organism, we show that exposure to polystyrene nanoplastics spanning 20–500 nm does not simply suppress motility, but redistributes organisms into distinct locomotor regimes characterized by altered exploration, reorientation dynamics, and behavioral phenotypes. In parallel, the polarization contrast associated with the digestive vesicles was markedly reduced and spatially associated with fluorescence‐validated nanoplastic accumulation. Orthogonal calcium imaging further reveals accompanying physiological perturbations. These findings establish polarization holographic 4D phenotyping as a scalable and sensitive platform for multiscale assessment of contaminant‐induced dysfunction in living aquatic organisms.