Phasor-FLIM visualizes and quantifies polluting nanoplastic particles in live organoids
Light Publishing Center, Changchun Institute of Optics, Fine Mechanics And Physics, CAS
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Figure | Principle of the phasor-FLIM barcoding approach and its application to studying the MNP interactions with small intestinal organoids. A, graphical representation of the approach. The polluting polymer is used for producing MNPs, labeled with the near-infrared dye, showing characteristic fluorescence lifetime. MNPs are exposed to the intestinal organoids (basal/apical-out…) with the polarity controlled by the live microscopy. Phasor-FLIM approach is used to analyze the uptake route, mechanism, physiological effect or quantifying the complex mixtures of the absorbed MNPs in the live organoid culture. B, confocal fluorescence images and corresponding phasor plots of organoids incubated with NPs (cyan), co-stained with Nile Red (yellow), demonstrate positive accumulation of only NP B and D, with the characteristic lifetimes confirmed by phasor-FLIM.
view moreCredit: Okkelman, I.A., Zhou, H., Borisov, S.M. et al.
Plastics pervade every aspect of the human life, being present in packaging and household goods, agricultural, food and medical products. Their resistance to (bio)degradation has resulted in the global accumulation of micro- and nanoplastics (MNPs) across diverse ecosystems, including soils, animals, aquatic environments, the atmosphere, food sources, and plants. Disturbingly, MNPs have already been detected in human blood, the brain, the gut, the reproductive system, and various other organs, raising pressing demand to understand their potential health effects. Recent studies with rodents and human cells already demonstrate the potential negative impact of MNPs on cell metabolism, oxidative stress and inflammation. However, it remains unclear whether these effects occur in human tissues or across diverse animal species within a physiologically relevant three-dimensional context. Therefore, to establish a fundamental understanding of MNP uptake, mechanisms and their effects on three-dimensional gastrointestinal tissue must be elucidated.
In a new paper published in Light: Science & Applications, a team of scientists, led by Professor Dmitriev from Tissue Engineering and Biomaterials Group, Ghent University (Ghent, Belgium), and co-workers, have developed a novel live imaging approach, addressing the dynamics of the MNP interaction with live intestinal tissue, within 3D and near-to-physiological setting: the team has designed ‘model’ near-infrared (NIR) emitting nanoplastic particles, which display characteristic differences in fluorescence lifetime (intrinsic characteristic for every luminescent molecule, reflecting its excited state duration), dependent on the type of the polymer material and the (micro)environment. The predicted and ‘pre-calibrated’ nanoplastic particles fluorescence lifetimes are highly amenable for phasor-FLIM ‘barcoding’ method, allowing for quantifying and separating different MNP types. Subsequently, the team applied this approach to probe MNP interactions with pig and mouse small intestinal organoid cultures, present in either basal-out or apical-out topology, to reveal the structure-activity relationships for some widely present polluting polymers. This methodology enables deeper understanding of the MNP interaction with tractable in vitro and potentially multi-organ-on-a-chip and in vivo models. The presented ‘lifetime barcoding’ feature allows for detecting different types of MNP and potentially their interactions with a non-plastic matter, e.g. ions, biomolecules, toxic chemicals or metals. After observing the positive and “MNP type”-specific accumulation, the team also demonstrated how the proposed imaging pipeline can be combined with the studies of the mitochondrial dynamics, polarization and expression of inflammatory cytokines. Collectively, the presented approach is expected to help studying post-internalization intracellular fate of the absorbed or endocytosed MNPs, across the broad range of physiologically relevant biological models, including organoids, animal tissues and beyond, bringing improved understanding of the biological effects of the MNPs to the human, animals, inter-species communities and potentially other members of the kingdom of life.
“We evaluated internalization of the nanoplastics into the apical-out and basal-out organoids and their concentration-dependent uptake. Strikingly, when compared with conventional intensity-based quantification, phasor FLIM-based event counting demonstrated significantly improved sensitivity and specificity. Importantly, we did not detect any pixel counts in the control ‘no NP’ control organoids (40 organoids analyzed in total in both experimental replicates R1 and R2) with the chosen analysis settings, which made a rare event count value more significant.”
“In addition, the presented phasor-FLIM ‘barcoding approach’ can discriminate between the different types of nanoplastics within same organoid. By setting the fingerprint zones for NP B (PMMA-MA) and NP D (PS-MA), we quantified the percentage of total cluster points in NP D, D/B and B lifetime zones, enabling accurate detection of MNP composition inside intestinal organoids when exposed to a complex mixture of MNPs.”
“Phasor-FLIM lifetime barcoding feature allows for multiplexed MNP detection and can be potentially applied to other fluorescent nanoparticles (e.g. ion-, biomolecule- or metal-related probes) to investigate their interaction with intestinal plasma membrane domain and their biological effect on gastrointestinal tissue across in vitro organoid, microphysiological on-a-chip platforms or in vivo live tissue models.” the scientists forecast.
Article Title
Visualizing the internalization and biological impact of nanoplastics in live intestinal organoids by Fluorescence Lifetime Imaging Microscopy (FLIM)
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