Whole-Tissue Distribution Analysis for Visualization of Nanoplastics in the Mouse Brain

Abstract Nanoplastics (NPs) are increasingly recognized as environmental contaminants and have been detected in diverse biological tissues; however, their biodistribution in complex organs remains poorly understood. Conventional section-based imaging restricts spatial context and volumetric analysis, making it challenging to map NP distribution in structurally complex organs such as the brain, even with fluorescent labeling. To address these limitations, we developed an integrated workflow combining tissue optical clearing (SeeDB2G) with light-sheet fluorescence microscopy (LSFM), enabling three-dimensional visualization of fluorescently labeled polystyrene (PS) NPs in neonatal mouse brains. At postnatal day 0 - a critical window of heightened vulnerability due to immature barrier systems and rapid neurodevelopment - pups were orally administered spherical PSNPs of 50 or 500 nm. Fluorescence stereoscopic imaging revealed pronounced organ-level accumulation of 50 nm PSNPs in the intestine, liver, kidney, and brain, compared with markedly lower signals from 500 nm PSNPs 24 h post-exposure. Optical clearing rendered the entire brain transparent while preserving fluorescence, allowing LSFM to accurately assess regional PSNP accumulation without sectioning. This workflow enabled whole-brain visualization of size-dependent NP uptake, with 50 nm PSNP detected throughout the brain and the highest accumulation observed in the thalamus and brainstem. Although the current implementation relies on fluorescent labeling and model NPs, this approach provides a scalable platform for whole-organ biodistribution analysis and lays the foundation for mechanistic studies of barrier permeability, developmental vulnerability, and organ-specific interactions. Competing Interest Statement The authors have declared no competing interest.