Raman and fluorescence together close a gap in microplastics detection

Research published in 2025 in the journal Environmental Monitoring and Assessment set out to answer a practical question for environmental laboratories: when two established microplastics detection methods disagree, which one should analysts trust?

Researchers at Trier University and the University of Birmingham compared confocal micro-Raman spectroscopy against Nile Red-assisted fluorescence microscopy, applying both to the same particle samples. The overall discrepancy in particle counts between the two methods reached 421 per cent, driven mainly by differences in how each technique handles particle size and shape rather than by the type of polymer involved.

Why the two methods disagree

The two methods have long been understood to have complementary strengths and weaknesses.

Nile Red fluorescence microscopy is fast and well suited to high sample throughput, but it can generate false positives when organic residues, often left behind by incomplete digestion with Fenton's reagent during sample preparation, fluoresce similarly to plastic particles.

Confocal micro-Raman spectroscopy is slower and more resource-intensive but distinguishes plastic from organic material far more reliably, because it identifies the specific chemical bonds present rather than relying on a fluorescent response.

Complementary techniques, not competing ones

The study's authors frame the two techniques as complementary rather than competing.

Using Raman analysis to validate a subset of particles flagged by Nile Red screening would let laboratories retain fluorescence's throughput advantage while correcting for its known blind spot around organic interference, rather than forcing a choice between speed and specificity.

Notably, the size-dependence of the discrepancy means the two methods do not simply disagree by a constant margin; smaller particles, which are harder to characterise definitively by either technique, appear to drive a disproportionate share of the mismatch, which has implications for any programme setting a minimum reportable particle size.

A significant gap for regulatory reporting

The finding lands at a useful moment for environmental laboratories, which face growing pressure to standardise microplastics reporting as regulators and standards bodies work toward harmonised methods.

A 421 per cent discrepancy between two established techniques is a significant gap for any laboratory reporting particle counts to a regulator, a client or a research programme expecting comparable data across sites or over time.

It also complicates comparisons between studies or monitoring programmes that have each relied on only one of the two methods, since neither their totals nor their size distributions can be assumed to be directly comparable, a problem that becomes more consequential as regulatory bodies increasingly attempt to compile national or cross-border microplastics datasets from work commissioned across multiple contract laboratories.

Instrumentation cost drives method choice

Method choice in this field has historically been driven as much by what instrumentation a laboratory already owns as by any settled view on which technique performs best for a given matrix.

Nile Red screening requires comparatively modest capital investment and can be run by technicians without extensive spectroscopy training, which has made it attractive to laboratories scaling up microplastics services quickly to meet client demand.

Confocal micro-Raman systems represent a larger capital outlay and a longer training curve, but this study adds to the case that laboratories offering microplastics analysis without any Raman validation step may be reporting counts with a degree of uncertainty their clients are not being told about.

For environmental laboratories under pressure to process high volumes of water, soil or air samples for microplastics, the finding is a case for combining rather than choosing between the two approaches, and a reminder that a single-method particle count, however efficiently generated, may not be a reliable one without a validation step behind it.

Laboratories quoting turnaround times and prices for microplastics work may also want to be explicit with clients about which method, or combination of methods, underpins the figures being reported, rather than leaving that detail implicit in a methods statement few clients read closely.