Visualization of Labelled Micro- and Nanoplastics in Interaction with Algae, using Super-Resolution STED Microscopy and Fluorescence Lifetime Imaging

Authors:

Marie Sioen, Herlinde De Keersmaecker, Maaike Vercauteren, Colin Janssen, Jana Asselman

Keywords:

Micro- and nanoplastics, Algae, Super Resolution Microscopy, autofluorescence, Fluorescence Lifetime Imaging

Abstract:

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 non-fluorescent micro and nanoplastics (MNPs) of various polymer types, shapes and sizes, in interaction with marine primary producers, which are autofluorescent. A labelling-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. While 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 (OM) 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.

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