BPS2026 – Visualizing the internalization and biological impact of nanoplastics in live organoid models using fluorescence lifetime imaging microscopy (FLIM)

Micro- and nanoplastic (MNP) pollution is a growing environmental challenge posing significant health risks for human and other living organisms. In particular, mechanisms of MNP accumulation and effects on absorptive tissues remain poorly understood. We address this knowledge gap using tractable models coupled to dynamic live-cell fluorescence imaging methods, enabling multi-parameter single-cell analysis. Here, we report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy (FLIM) to study MNP interactions with intestinal epithelium. To facilitate this, we first optimized live imaging of porcine and mouse small intestinal organoids with an “apical-out” topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) polymers and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake. Using a phasor analysis approach, we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy. The resulting “fluorescence lifetime barcoding” enabled distinguishing of different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total cell energy budget and epithelial inflammation. We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in other live organoid and 3D ex vivo tissue systems. Funding: supported by the European Union, fliMAGIN3D-DN Horizon Europe-MSCA-DN grant no. 101073507.