Multimodal imaging and quantification of lanthanide chelate-labeled micro- and nanoplastics in plants

Understanding the uptake and distribution of microplastics (MPs) and nanoplastics (NPs) in plants is crucial for assessing their environmental impact. This can be effectively achieved using labeled model particles. While conventional fluorophores are limited by plant autofluorescence, photobleaching, and poor quantitative resolution, in our work we incorporate stable lanthanide (europium) chelates, enabling long-lifetime luminescence detection that effectively eliminates this background interference. Here this protocol integrates time-gated fluorescence imaging microscopy, electron microscopy coupled with energy-dispersive X-ray spectroscopy, and inductively coupled plasma mass spectrometry. The solvent swelling labeling method is exemplified using MPs/NPs with different chemical compositions and shapes. For comparison, procedures using MPs/NPs labeled with conventional fluorescent dyes, such as Nile blue chloride (red fluorescence) and the amine-reactive probe 4-chloro-7-nitro-2,1,3-benzoxadiazole (for green fluorescence) are included. The workflow follows a tiered approach: time-gated fluorescence imaging microscopy rapidly localizes particles within tissues, electron microscopy coupled with energy-dispersive X-ray spectroscopy provides subcellular verification and elemental confirmation, and inductively coupled plasma mass spectrometry quantifies total particle accumulation with high sensitivity. This provides a robust framework for mechanistic studies of MPs/NPs uptake, translocation and partitioning from the subcellular to the whole-plant level. The protocol requires moderate expertise in microscopy and analytical chemistry and takes 2-4 months to complete, depending on the plant species (procedures for hydroponic and soil-grown wheat and lettuce are provided). It is designed specifically for hypothesis-driven research using well-defined, labeled model particles and is not intended for the environmental monitoring of unlabeled plastics in field samples.