Visualising Microplastics incorporated into Corals: A Rapid and Direct Chemical Imaging Method
1. Key Points
- A key to tracing the history of marine plastic pollution
Since corals form growth bands as they develop their skeletons, they have the potential to be used to trace back a history of microplastic fragments previously incorporated by identifying their location in the skeletal matrix. However, as conventional methods for plastic identification require long measurement times, they are not realistically applicable to scanning the coral skeleton. Using the proposed method, microplastics can potentially be identified within the skeleton, which will contribute to reconstructing the historical progression of plastic pollution. - Rapid and direct visualisation of microplastics in corals
A chemical analytical method for the rapid and direct visualisation of microplastics incorporated in corals has been established by combining coherent anti-Stokes Raman scattering (CARS)※1 and two-photon excited fluorescence (TPEF)※2 techniques. By scanning the surfaces of coral skeletons and tissue at speeds significantly faster than conventional methods, micrometre-sized plastic particles have been successfully visualised and quantified while clearly distinguishing plastic signals from the coral’s biological structure without the need for labels or dyes. - Correlation between coral health and plastic accumulation
Using the proposed method, the relationship between coral health and microplastic accumulation has been studied. Through the analysis of coral tissue and skeletons exposed to polyethylene (PE) beads in high concentrations, corals in poor health, exhibiting signs such as tissue loss or bleaching, were found to incorporate more microplastics into their tissue, as previously reported, and notably further into their skeletons than healthy ones. This implies that environmental stressors that cause coral bleaching, such as rising sea temperatures, may further accelerate the accumulation of microplastics within reef ecosystems.
Coherent anti-Stokes Raman scattering (CARS)
A label-free molecular analysis technique that detects signals from molecular vibrations. By simultaneously irradiating a pair of laser beams with a frequency difference matching the target molecular vibration, it generates significantly strong and chemically specific signals through vibrational coherence.
Two-photon excited fluorescence (TPEF)
A technique that generates fluorescence by simultaneously absorbing two photons. It is generated using the same laser light source as CARS but emits at different wavelengths, allowing for the simultaneous and separate acquisition of signals.
Fig. 1 Conceptual diagram of this research.
2. Overview
Dr. Tomoko Takahashi at the Japan Agency for Marine-Earth Science and Technology (JAMSTEC), in collaboration with the Biophotonics group at the University of Southampton, has successfully established a chemical imaging technique to rapidly and directly visualise microplastics incorporated into corals.
Coral skeletons form growth bands as they develop, implying that if microplastics are trapped within this skeletal matrix, their position can provide a chronological archive of pollution. While in laboratory settings, experiments often use fluorescent-labelled or easily recognisable microplastics, plastics in the natural environment typically lack such distinctive features. Conventional analytical methods that can identify such generic microplastic materials, such as FT-IR or Raman spectroscopy※3, are not practically applicable to scanning large areas of coral skeletons to map incorporated microplastic fragments due to their fundamentally long measurement times per spectral acquisition.
To address these challenges, the team applied CARS and TPEF combined microscopy, a multimodal technique typically used for imaging components in biomedical samples such as cells, to achieve high-resolution imaging of PE beads incorporated in corals. This method allows target molecules to be clearly distinguished from the coral structures (i.e., skeletons and tissue), enabling direct and rapid mapping without adding labels. The study indicates that while healthy corals show minimal plastic uptake, corals with tissue loss show concentrated accumulation in their tissue, consistent with previous reports, and notably also within their skeletons (Figure 2). This finding demonstrates a proof of concept of a methodology that could make it possible to use corals as an “environmental archive” for marine plastic pollution.ト
This work was supported by the Japan Society for the Promotion of Science (JSPS) (20KK0336, 21H01557), the Natural Environment Research Council (NE/T001364/1), European Research Council Advanced Grant Microns2Reefs (884650), Engineering and Physical Sciences Research Council Grant (EP/T020997/1), and the University of Southampton (DTP EP/N509747/1).
Tomoko Takahashi1,2, Cecilia D'angelo2, Jacob Kleboe2, Joerg Wiedenmann2, Gavin L. Foster2, Sumeet Mahajan2
- JAMSTEC
- University of Southampton
FT-IR and Raman Spectroscopy
Analytical methods used to identify compounds based on the interaction between light and molecular vibrations. FT-IR spectroscopy utilizes infrared light absorption, while Raman spectroscopy uses laser scattering light. Since they are sensitive to different types of molecular vibrations, they provide complementary information.
Fig. 2 CARS and TPEF multimodal imaging of coral skeletons. PE beads (red, CARS signal) are clearly visualised as they are incorporated into the coral skeleton (green, TPEF signal). CS: cross section, scale bar: 5 µm.
3. Background
Marine plastic pollution has become a serious global threat. In particular, microplastics with sizes less than 5mm are detected not only in water and sediments, but also in the bodies of large- to small-scale marine organisms, and their impacts are a matter of serious concern. Among these organisms, reef-building corals have interesting characteristics that could keep incorporated microplastics in their skeletons with growth bands, and this offers the possibility to provide a record of microplastic exposure over time.
While laboratory studies often utilise fluorescent-labelled plastic particles, microplastics found in the natural environment are typically less distinctive. To identify these generic plastics, analytical techniques that detect signals from molecular vibrations, such as FT-IR or Raman spectroscopy, are commonly used. However, these methods require long measurement times, typically several seconds to minutes per spectral acquisition at one point, making it difficult to scan the large skeletal areas required for tracing pollution history. While quantitative analysis of incorporated microplastics in corals can be performed through destructive techniques that require dissolution of the skeleton, this results in the loss of spatial information relating to the location and timing of plastic incorporation. Therefore, a rapid, non-destructive analytical technique is required to develop a coral-based archive of marine plastic pollution.
4. Research results
- Direct 3D visualisation of microplastics using CARS and TPEF combined microscopy
CARS microscopy generates strong signals due to vibrational coherence, allowing for rapid signal acquisition. By simultaneously and separately capturing TPEF signals from the coral skeleton and CARS signals from the plastics, both components can be clearly distinguished and mapped directly (Figure 2). This multimodal approach enables significantly faster imaging (less than 2 seconds for a 424 µm×424 µm image with 0.4 µm resolution) than conventional methods, making wide-area 3D visualisation of microplastics within the corals possible. - Incorporation into coral skeleton and tissue
Analysis of corals exposed to PE particles with high concentrations in a laboratory setting clearly visualised micrometre-sized particles incorporated near the skeletal surface (Figure 2). These particles were identified without the use of dyes or labels, demonstrating that the process of microplastic incorporation alongside skeletal growth can be captured with high spatial resolution (sub-micron level). - Correlation between coral health and plastic incorporation
Quantitative analysis found that microplastics were rarely detected in healthy tissue, whereas corals showing signs of bleaching or tissue loss tend to accumulate microplastics in their tissue (Figure 3), which is consistent with previous studies, and also in their skeletons. This demonstrates that unhealthy conditions could accelerate microplastic incorporation into coral tissue and further into their skeletons.
Fig. 3 CARS and TPEF multimodal imaging of coral tissue. (a-b) Tissue with partial tissue loss, (c-d) healthy tissue. PE beads are highlighted in red; coral tissue structures are highlighted in pink and green. PE beads (red) are detected only in (a-b) tissue where bleaching/tissue loss was observed. Scale bar: 100 µm.
5. Future outlook
The method proposed in this study is applicable to various types of microplastics and is expected to be applied to coral samples collected from natural environments. By linking the growth bands of coral skeletons with the position of incorporated plastics, it will be possible to reconstruct a detailed timeline of marine pollution.
Furthermore, the finding that corals with tissue loss tend to accumulate microplastics in their tissue and further into their skeletons suggests that environmental changes causing bleaching, such as rising sea temperatures, may accelerate microplastic incorporation into corals. This indicates that the combined stress of environmental change and plastic pollution could have an impact on coral reef ecosystems, and this technique will contribute to monitoring these long-term environmental risks.
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