Rapid methods for the quantification of ingested nano-and microplastics in marine fish by imaging flow cytometry

Quantitative analysis of nano- and microplastics (NMPs) in biota samples has been hindered by stringent and evolving technical requirements. And conventional analytical techniques are typically constrained by inherent micrometer-scale detection limits and low-throughput workflows, thus limiting their applicability for the accurate and rapid enumeration of nanoplastics (NPs). In this study, a high-throughput analytical method for the quantitative analysis of microplastics (MPs, 3 µm polystyrene (PS) spheres in biota samples was adapted and optimized by imaging flow cytometry (IFC). Systematic optimization demonstrated that 1 µL·mL Tween 20 efficiently disperses target particles, while 10 µL·mL Nile Red delivers sufficient fluorescence intensity without excessive background interference. An acquisition volume of 50000 particles ensures maximal analytical stability and data reliability. Potential biological matrix interference was effectively mitigated via appropriately extending the digestion period and reducing the amount of tissue used, thereby minimizing the content of residual organic matter. Finally, a calibration curve incorporating a morphology-based correction factor effectively compensated for particle-aggregation bias, significantly improving quantitative accuracy. This method was validated through controlled exposure experiments in fish, demonstrating both feasibility and accuracy providing robust technological support for ecological risk assessments of NMPs.