Visualization of labeled micro- and nanoplastics in interaction with algae, using super-resolution stimulated emission depletion microscopy and fluorescence lifetime imaging.

In contrast to microplastics, studying the interactions of nanoplastics (NPs) with primary producers such as marine microalgae remains challenging. This is attributed to the lack of adequate visualization methods that can distinguish NPs from autofluorescent biological material such as marine algae. The aim of this study was to develop a method for labeling and visualizing nonfluorescent micro- and nanoplastics (MNPs) of various polymer types, shapes, and sizes, in interaction with marine primary producers, which are autofluorescent. A labeling technique for plastics was refined, using a swell incorporation method with the commercial dye "IDye." Comprehensive quality control measures, including toxicity, leaching, and dye longevity tests, were applied to ensure the robustness of the method. Although stimulated emission depletion (STED) microscopy successfully enabled the visualization of the diverse labeled NPs smaller than 200 nm, it could not distinguish NPs from autofluorescent organic material such as marine microalgae, due to overlapping excitation and emission spectra with the photosynthetically active molecule chlorophyll-a. This study is the first to advance the field by coupling STED with fluorescence lifetime imaging microscopy (FLIM). The FLIM technique, based on the differing lifetimes of fluorescent signals, allowed us to overcome the challenge of overlapping spectra. Our work not only refines and expands existing plastic labeling protocols to accommodate a wide range of polymer types, but also introduces a more precise method for studying interactions between MNPs and autofluorescent organisms. This combined STED-FLIM approach provides a reproducible and reliable framework for examining MNP impacts in complex, ecologically relevant environments, particularly highlighting its potential for investigating MNP-microalgae interactions.