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Label-free 4D confocal Raman imaging enabling in vivo multiplex identification of microplastics in zebrafish
He Zhu1, Jing Luo2, Sailing He3
1Centre for Optical and Electromagnetic Research, National Engineering Research Center for Optical Instruments, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310058, China.
Abstract:
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.
Insights
This study introduces a label-free imaging system to track multiple unlabeled microplastics (MPs) in live zebrafish. The novel method distinguishes different polymer types and monitors their accumulation and excretion, offering insights into plastic pollution dynamics.
Area of Science:
- Environmental Science
- Biotechnology
- Analytical Chemistry
Background:
- Microplastics (MPs) in nature are complex mixtures.
- Existing in vivo imaging struggles with simultaneous tracking of multiple, unlabeled MPs.
- Invasive labeling methods can alter MP behavior and biological interactions.
Purpose of the Study:
- To develop a label-free imaging system for simultaneous, in vivo identification of multiple microplastic types.
- To investigate the ingestion, accumulation, and excretion dynamics of mixed microplastics in live organisms.
- To provide a novel analytical tool for assessing microplastic bioaccumulation.
Main Methods:
- Developed a four-dimensional confocal Raman imaging (4D-cRI) system with high spatial resolution and broad wavenumber coverage.
- Utilized label-free chemical specificity to image both microplastics and endogenous biomolecules in live zebrafish.
- Quantified microplastic distribution and abundance using Raman-positive voxel counts.
Main Results:
- Successfully achieved multiplex identification and in vivo discrimination of five common microplastic types (PA, PE, PS, PET, PMMA) in zebrafish.
- Visualized distinct biomolecular signals alongside ingested microplastics.
- Demonstrated dynamic monitoring of microplastic ingestion, gut accumulation (significant after 8h), and excretion.
- Revealed differential retention behaviors among various polymer types.
Conclusions:
- The 4D-cRI system offers a robust, high-precision method for analyzing polymer-specific ingestion and in vivo accumulation of mixed microplastic pollutants.
- This technology provides a powerful tool for evaluating microplastic bioaccumulation dynamics in complex biological systems.
- The findings highlight the potential for distinct accumulation patterns of different microplastic types within organisms.
