Scanning zebrafish for microplastics: Micro-CT as a non-destructive way to image microplastics in biological samples

Plastic pollution is now recognized as a global crisis due to its persistence and widespread distribution in the environment. While macroplastic pollution has long been a concern, microplastics – plastic fragments ranging from 1 to 1000 µm – pose an increasingly serious threat to ecosystems as their proliferation in the food chain could be potentially dangerous to human health. A newly published study by Viktória Parobková from CEITEC Brno University of Technology and co-authors presents the use of micro-CT to image microplastics without damaging the biological sample. The study is a proof of concept and demonstrates the benefits of the methodology on zebrafish (Danio rerio), with potential applications to other biological samples.

Traditional methods such as spectroscopy and pyrolysis have been successfully applied to detect microplastics (MPs) in biological samples, primarily to assess their impact on organisms. However, these techniques come with significant limitations, particularly regarding sample preparation. Digestion or slicing of tissues is often required, which introduces two significant challenges: the loss of spatial distribution information and an increased risk of contamination, potentially leading to false positives.

In the Laboratory of X-ray micro and nano computed tomography at CEITEC BUT, the research team employed advanced micro-CT systems that overcome these drawbacks by allowing direct analysis of biological samples without destructive preparation. This preserves critical spatial information, enabling us to identify MP localization and accumulation sites – key factors in understanding potential toxicological effects. The application of micro-CT on biological samples, in this case zebrafish (Danio rerio), a popular aquarium fish, is the subject of the Advancing microplastic detection in zebrafish with micro-computed tomography: A novel approach to revealing microplastic distribution in organisms study published in the Journal of Hazardous Materials.

Why is the spatial distribution of microplastic particles important? Understanding where MPs accumulate in tissues could provide vital insights into their role in health issues. For instance, previous studies have shown that prolonged MP exposure can cause significant damage to the small intestine of fish, affecting their digestion, growth, and reproduction. When studying the broader effects of MPs in the gastrointestinal tract, micro-CT can provide a non-destructive, high-resolution imaging approach.

With all the advantages of micro-CT, there is one drawback that was challenging for the team to overcome. Since micro-CT depends on density differences to visualise structures and MPs typically have low density, it was difficult to distinguish them from surrounding biological tissues. To solve this problem, the team increased the contrast between MPs and tissues by employing specific staining techniques. Polyethylene microplastics were used as reference particles since they are known type, size, and shape. When combined with iodine staining, the selected polyethylene MPs indeed exhibited sufficient contrast against surrounding tissue, which is essential for soft tissue visualization in micro-CT.

Another key factor in successful detection was spatial resolution. The team had to balance the need for high resolution with their efforts to visualise the complexity of the sample. While smaller samples yield higher spatial resolution, their smaller size also means that they only show a limited region of the body. “To balance the two approaches, we have developed two scanning strategies: whole-fish scans that focus on the abdominal region and extracted gut scans that provide finer detail due to the reduced sample volume. This trade-off between sample size and resolution is critical when designing micro-CT-based microplastic detection methodologies,” explains Viktória Parobková.

The study serves as a proof of concept demonstrating, for the first time, the feasibility of using micro-CT to detect microplastics in biological tissues. “Given that we have worked with well-defined PE MPs, our next steps will focus on expanding the technique to different MP types, shapes, and also densities – different polymers exhibit distinct densities, affecting how well they can be visualized using micro-CT,” Viktória says. By systematically exploring these variables, the team aims to refine the methodology and assess its broader applicability for environmental and toxicological studies.

The methodology was developed using zebrafish but it is adaptable to other biological models. Micro-CT, therefore, holds promise for biomedical applications, particularly in assessing MP accumulation in digestive tissues. The main drawback is that MPs would likely need to be at the nanometer scale to cross cellular barriers and be detectable in the systemic circulation, which is a significant challenge for micro-CT with current technology. However, with advancements in resolution and imaging techniques, future micro-CT systems may be capable of detecting even these smallest particles. This methodology could thus one day contribute to studies on the potential health risks of microplastic exposure in humans.

Source: CEITEC BUT

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