Evaluating Microplastic Detection with Fluorescence Microscopy and Raman Spectroscopy

A recent study led by researcher Steve Utecht of the University Trier, explored a new approach that could help improve the accuracy of microplastic detection in the environment. This study evaluated two techniques: confocal micro-Raman spectroscopy and Nile Red-assisted fluorescence microscopy (1).

What are microplastics?

Microplastics are miniature pieces of plastic that are 5 millimeters or smaller (1,2). They are ubiquitous in the environment because of anthropogenic activities such as manufacturing (2). Although scientists are aware of their pervasiveness in the environment, there is not much consensus on what analytical methods are best for characterizing these plastics in the environment. Previous research efforts have showed that spectroscopy techniques, such as Raman spectroscopy, can be used in this effort (2,3).

What did the researchers test in this study?

In this study, Utecht and his team developed a comparative study of 100 environmental samples and applied both confocal micro-Raman spectroscopy and Nile Red-assisted fluorescence microscopy to each sample both independently and in combination (1). The main objective was to test whether both methods performed better individually or with one another.

What were the results of the study?

The authors found that high variability existed in the detection outcomes between the two methods, which is not uncommon in microplastic analysis (1). The overall percentage difference in results between confocal micro-Raman spectroscopy and Nile Red-assisted fluorescence microscopy was 421%, with discrepancies varying by three orders of magnitude (1). The differences were influenced most significantly by the size and morphology of the microplastics (for example, particles versus fibers), rather than the type of plastic polymer (1).

The researchers also found that both methods showed improved agreement when analyzing smaller-sized particles (1). This finding indicates that smaller MPs are more reliably detected across different analytical platforms, possibly because of their more uniform morphology and less likelihood of being obscured by residual organic matter (1). In contrast, the techniques yielded greater discrepancies when larger particles (>75 µm) or fibrous materials were examined (1).

What were the limitations of Nile Red-assisted fluorescence microscopy?

For Nile Red-assisted fluorescence microscopy, the researchers found that one limitation of the technique was that it was susceptible to false positives (1). Nile Red-assisted fluorescence microscopy uses Nile Red dye to fluoresce when bound to hydrophobic surfaces. The problem with this is that the dye can bind to natural organic residues, which makes it difficult to use when organic matter is not removed from samples completely (1). As a result, the authors acknowledge that this could lead to inaccurate and inflated microplastic counts (1).

That is why the researchers also tested combining this technique with confocal micro-Raman spectroscopy. Because Raman spectroscopy offers higher specificity, it can differentiate microplastics from organic materials based on their molecular vibrations (1). As a result, this dual-method strategy leverages the speed and throughput of Nile Red-assisted fluorescence microscopy with the specificity of confocal micro-Raman spectroscopy to build an improved detection protocol.

What are the key takeaways from the study?

One key takeaway from this study is that better understanding of how sample preparation could affect microplastics before analysis is important. Smaller microplastics (<125 µm) may degrade or change shape because of solvent exposure, further complicating accurate identification and quantification (1).

The research team emphasized that future studies should continue to refine organic matter removal techniques and explore solvent effects on MP stability (1). Additionally, developing standard operating procedures that integrate multiple detection methods will be essential for producing consistent, accurate data across laboratories and regions.

References

1. Utecht, S.; Krause, S.; Schuetz, T. Quantitative and Qualitative Differences of Common Microplastic Detection Procedures: Nile Red-assisted Fluorescence Microscopy and Confocal Micro-Raman Spectroscopy. Research Square 2025, ASAP. DOI: 10.21203/rs.3.rs-5803470/v1
2. Wetzel, W. Measuring Microplastics in Remote and Pristine Environments. Spectroscopy. Available at: https://www.spectroscopyonline.com/view/measuring-microplastics-in-remote-and-pristine-environments (accessed 2025-07-01).
3. Wetzel, W. Quantifying Microplastics and Anthropogenic Particles in Marine and Aquatic Environments. Spectroscopy. Available at: https://www.spectroscopyonline.com/view/quantifying-microplastics-and-anthropogenic-particles-in-marine-and-aquatic-environments (accessed 2025-07-01).