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Dark-field/hyperspectral microscopy for detecting nanoscale particles in environmental nanotoxicology research
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Researchers reviewed enhanced dark-field microscopy combined with hyperspectral imaging as a non-destructive optical technique for detecting and chemically characterizing nanoscale particles — including nanoplastics — in live, wet biological samples where electron microscopy is impractical.
Nanoscale contaminants (including engineered nanoparticles and nanoplastics) pose a significant threat to organisms and environment. Rapid and non-destructive detection and identification of nanosized materials in cells, tissues and organisms is still challenging, although a number of conventional methods exist. These approaches for nanoparticles imaging and characterisation both inside the cytoplasm and on the cell or tissue outer surfaces, such as electron or scanning probe microscopies, are unquestionably potent tools, having excellent resolution and supplemented with chemical analysis capabilities. However, imaging and detection of nanomaterials in situ, in wet unfixed and even live samples, such as living isolated cells, microorganisms, protozoans and miniature invertebrates using electron microscopy is practically impossible, because of the elaborate sample preparation requiring chemical fixation, contrast staining, matrix embedding and exposure into vacuum. Atomic force microscopy, in several cases, can be used for imaging and mechanical analysis of live cells and organisms under ambient conditions, however this technique allows for investigation of surfaces. Therefore, a different approach allowing for imaging and differentiation of nanoscale particles in wet samples is required. Dark-field microscopy as an optical microscopy technique has been popular among researchers, mostly for imaging relatively large specimens. In recent years, the so-called "enhanced dark field" microscopy based on using higher numerical aperture light condensers and variable numerical aperture objectives has emegred, which allows for imaging of nanoscale particles (starting from 5 nm nanospheres) using almost conventional optical microscopy methodology. Hyperspectral imaging can turn a dark-field optical microscope into a powerful chemical characterisation tool. As a result, this technique is becoming popular in environmental nanotoxicology studies. In this Review Article we introduce the reader into the methodology of enhanced dark-field and dark-field-based hyperspectral microscopy, covering the most important advances in this rapidly-expanding area of environmental nanotoxicology.
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Detection of unlabeled nanoplastics within Daphnia magna using enhanced darkfield hyperspectral microscopy
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Researchers developed a method to detect unlabeled nanoplastics within Daphnia magna using enhanced darkfield hyperspectral microscopy combined with a post-exposure histological labeling process. The study presents a new approach for identifying nanoplastic particles in model organisms without requiring pre-labeled or fluorescent plastics.
Dark-field hyperspectral microscopy for label-free microplastics and nanoplastics detection and identification in vivo: A Caenorhabditis elegans study
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Researchers demonstrated that dark-field hyperspectral microscopy can visualize and chemically identify nano- and microplastics (down to 100 nm) in live C. elegans nematodes without labeling, differentiating multiple polymer types simultaneously within intestinal tissue.
Accumulation of nanoplastics in human cells as visualized and quantified by hyperspectral imaging with enhanced dark-field microscopy
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Researchers developed a label-free imaging technique to visualize and count nanoplastic particles that accumulate inside human cells, using enhanced dark-field microscopy combined with hyperspectral imaging. The method successfully tracked polystyrene nanoplastics entering cells over time and measured accumulation rates without needing fluorescent labels. This tool could improve the accuracy of future studies assessing how nanoplastics build up in human tissue and what concentration levels may pose health risks.
Fluorescence-Free Tracking of Polystyrene Microplastics in Mosquito Larvae Using Dark-Field Hyperspectral Microscopy
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Researchers used dark-field hyperspectral microscopy to track the distribution of 2-micrometer polystyrene microspheres in mosquito larvae without relying on fluorescence, finding that histological processing quenches fluorescence and alters spectral properties, and demonstrating that enhanced dark-field microscopy coupled with hyperspectral imaging is a reliable fluorescence-free method for mapping microplastic biodistribution in complex tissues.
Label-free detection of micro- and nanoplastics using dark-field hyperspectral and atomic force microscopies
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Researchers demonstrated that dark-field hyperspectral microscopy could visualize and spectrally identify polystyrene, polymethacrylate, and melamine formaldehyde particles (100 nm to 2 µm) in living Caenorhabditis elegans in vivo, while atomic force microscopy in PeakForce Tapping mode resolved internalized versus surface-bound polystyrene particles (down to 500 nm) in human skin fibroblasts. Deep learning algorithms applied to enhanced dark-field images further enabled automated classification of pigmented microplastics, demonstrating a label-free toolkit for detecting and differentiating particles in biological systems.
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