Label-free 4D confocal Raman imaging enabling in vivo multiplex identification of microplastics in zebrafish

Microplastics (MPs) in natural environments typically exist as heterogeneous mixtures of various polymer types. However, current in vivo imaging techniques are often limited to single-component tracking or involve invasive fluorescent labeling, which fail to support the simultaneous identification of multiple unlabeled microplastics in vivo. In this study, we address this challenge by developing a label-free four-dimensional confocal Raman imaging (4D-cRI) system. It features a high spatial resolution (∼500 nm) and a broad wavenumber coverage (500-3200 cm⁻¹). It enables chemically specific imaging of both exogenous microplastics and endogenous biological molecules such as lipids and proteins in live zebrafish. We visualized the distribution patterns of intrinsic biomolecular signals in zebrafish, and further validated the multiplex identification capability for five common microplastics (PA, PE, PS, PET, and PMMA), achieving in vivo discrimination of the ingested polymer types in live zebrafish exposed to this five-polymer mixture. Furthermore, we utilized the counts of detected Raman-positive voxels to evaluate relative particle abundance and dynamically monitor the ingestion, accumulation, and excretion processes of these mixed MPs. The results revealed distinct retention behaviors among different polymer types, and microplastics showed significant accumulation in the gut after 8 h of exposure. This work establishes a robust and high-precision methodology for analyzing polymer-specific ingestion differences and in vivo accumulation of mixed plastic pollutants, which provides a powerful analytical tool for evaluating microplastic bioaccumulation dynamics.