Accumulation of nanoplastics in human cells as visualized and quantified by hyperspectral imaging with enhanced dark-field microscopy

[Display omitted] •HSI-DFM can visualize and quantify the cellular accumulation of NPs.•The hyperspectral library was constructed and optimized based on intracellular NPs.•The accuracy of HSI-DFM was verified by fluorescence microscopy and flow cytometry.•HSI-DFM can determine the accumulation kinetics of NPs in single living cells.•HSI-DFM can also be applied to different cell lines and different NPs. Nanoplastic (NP) pollution is receiving increasing attention regarding its potential effects on human health. The identification and quantification of intracellular NPs are prerequisites for an accurate risk assessment, but appropriate methods are lacking. Here we present a label-free technique to simultaneously visualize and quantify the bioaccumulation of NPs based on hyperspectral imaging with enhanced dark-field microscopy (HSI-DFM). Using polystyrene NPs (PS NPs) as representative particles, the construction of a hyperspectral library was optimized first with more accurate NP identification achieved when the library was based on intracellular instead of extracellular PS NPs. The PS NPs used herein were labeled with a green fluorescent dye so that the accuracy of HSI-DFM in identifying and quantifying intracellular NPs can be evaluated, by comparing the results with those obtained by fluorescence microscopy and flow cytometry. The validation of HSI-DFM for use in determinations of the NP concentration at the single-cell level allows analyses of the accumulation kinetics of NPs in single living cells. The utility of HSI-DFM in different cell lines and with NPs differing in their chemical composition was also demonstrated. HSI-DFM therefore provides a new approach to studies of the accumulation and distribution of NPs in human cells.