Single-Particle Resolution Fluorescence Microscopy of Nanoplastics

Understanding of nanoplastic prevalence and toxicology is limited by imagingchallenges resulting from their small size. Fluorescence microscopy is widely applied to track andidentify microplastics in laboratory studies and environmental samples. However, conventionalfluorescence microscopy, due to diffraction, lacks the resolution to precisely localize nanoplasticsin tissues, distinguish them from free dye, or quantify them in environmental samples. To addressthese limitations, we developed techniques to label nanoplastics for imaging with stimulatedemission depletion (STED) microscopy to achieve resolution at an order of magnitude superior toconventionalfluorescence microscopy. These techniques include (1) passive sorption; (2) swellincorporation; and (3) covalent coupling of STED-compatiblefluorescence dyes to nanoplastics.We demonstrate that our labeling techniques, combined with STED microscopy, can be used toresolve nanoplastics of different shapes and compositions as small as 50 nm. The longevity of dye labeling is demonstrated indifferent media and conditions of biological and environmental relevance. We also test STED imaging of nanoplastics in exposureexperiments with the model wormCaenorhabditis elegans. Our work shows the value of the method for detection and localization ofnanoplastics as small as 50 nm in a whole animal without disruption of the tissue. These techniques will allow more preciselocalization and quantification of nanoplastics in complex matrices such as biological tissues in exposure studies