Single-Particle Resolution Fluorescence Microscopy of Nanoplastics

[Image: see text] Understanding of nanoplastic prevalence and toxicology is limited by imaging challenges resulting from their small size. Fluorescence microscopy is widely applied to track and identify microplastics in laboratory studies and environmental samples. However, conventional fluorescence microscopy, due to diffraction, lacks the resolution to precisely localize nanoplastics in tissues, distinguish them from free dye, or quantify them in environmental samples. To address these limitations, we developed techniques to label nanoplastics for imaging with stimulated emission depletion (STED) microscopy to achieve resolution at an order of magnitude superior to conventional fluorescence microscopy. These techniques include (1) passive sorption; (2) swell incorporation; and (3) covalent coupling of STED-compatible fluorescence dyes to nanoplastics. We demonstrate that our labeling techniques, combined with STED microscopy, can be used to resolve nanoplastics of different shapes and compositions as small as 50 nm. The longevity of dye labeling is demonstrated in different media and conditions of biological and environmental relevance. We also test STED imaging of nanoplastics in exposure experiments with the model worm Caenorhabditis elegans. Our work shows the value of the method for detection and localization of nanoplastics as small as 50 nm in a whole animal without disruption of the tissue. These techniques will allow more precise localization and quantification of nanoplastics in complex matrices such as biological tissues in exposure studies.