Combatting microplastics with microscopy

Imagine looking at a bowl of flour and trying to pick out a single granule of starch, would you be able to see it with the naked eye? Unless you’re a superhero, this would be impossible. But, of course, magnified visual or photographic images of objects can be produced using microscopes.

The ancient Egyptians were the first people known to utilise the concept of microscopy by using chips of crystal or obsidian to view small objects. Many millenniums later in Rome, gemstones were used by Emperor Nero to observe actors on a distant stage. Sometime during the 13th century, English philosopher Roger Bacon designed the first magnifier for scientific purposes.

These early light microscopes developed into simple (single lens) or compound (multiple lenses) microscopes, with the first electron microscopes designed and built in the 1930s, and confocal microscopes devised soon after. The versatility of microscopes and what they can image has contributed to many advances in science, broadening their relevance across diverse disciplines, with one being the detection of microplastics.

Microplastics are a huge environmental concern as they do not breakdown into harmless molecules and can take hundreds and thousands of years to decompose

Microplastics are defined as small particles or fragments of plastic measuring less than 5 mm in length, commonly originating from commercial product development or the breakdown of larger plastics. These are a huge environmental concern as they do not break down into harmless molecules and can take hundreds and thousands of years to decompose. They are especially dangerous for aquatic and marine life, due to their insolubility in aqueous environments.

The use of microscopic techniques to identify and characterise microplastics is gaining traction. By way of example, atomic force microscopy is a technique that produces images by measuring the forces between a sharp probe and the surface of a sample, providing a detailed topographical map. Previously, the degradation process of microplastics was observed using atomic force microscopy in the marine environment, particularly in the presence of bacteria adhered to the microplastics’ surface. However, atomic force microscopy has its limitations, suggesting it should be used in conjunction with other characterisation techniques such as spectroscopy.

Another system that is better when used alongside other characterisation techniques is stereomicroscopy, which provides 3D analysis of samples by observing from two different angles, making it a very effective and widely used technique. The images provide detailed information on the objects surface and structure, delivering a fast first-screening method of the shape, size and colour of microparticles. However, there are limitations to stereomicroscopy, including the difficulty characterising microplastics that are transparent or smaller than 100 µm.

Fluorescence microscopes form images by collecting the fluorescence emission from samples that have been excited by specific wavelengths, rendering fluorescence microscopy effective in detecting transparent or white microplastics. However, there are also limitations – microplastics often have chemical additives in their synthesis which can influence their fluorescent properties. A technique being explored to overcome this limitation is dying the samples with a fluorescent dye which labels the plastic fragments.

Microscopy can identify 20-70% of the total microplastics in a sample, so the techniques currently employed have limitations. For instance, the analysis of very small particles (<100 µm) in certain matrices is a challenge and requires the microscopy technique to be coupled with other analytical techniques

While the techniques mentioned are highly effective for analysing the surface characteristics of microplastics, they fall short in providing insights at the atomic scale. This is where techniques like electron microscopy shine through. Transmission electron microscopy forms images based on the electrons from an electron beam that are transmitted through an ultra-thin sample, revealing its internal structures at the atomic or molecular level. Microplastics are electron dense and must be stained with heavy-metals before imaging. This technique is more often used to study the effect of microplastics on model systems.

Whereas, scanning electron microscopy can be used in environmental mode to visualise microplastics in various matrices including sewage sludge, mussels, sediments and sand. In scanning electron microscopy, a beam of electrons scans the sample, producing secondary electrons that are detected giving information on the surface topography and composition of the sample.

Microscopy can identify 20-70% of the total microplastics in a sample¹, so the techniques currently employed have limitations. For instance, the analysis of very small particles (<100 µm) in certain matrices is a challenge and require the microscopy technique to be coupled with other analytical techniques like spectroscopy (i.e. FTIR and Raman). The presence of other small molecules or species within marine samples can make accurate identification of the microplastics difficult.

Emerging techniques like holographic imaging could overcome these limitations. This entails using a holographic microscope, which captures visual information by utilising lasers, with the holographic recording performed by a sensor. This technique is accurate enough to distinguish microplastics from organisms such as microplankton and microalgae in marine samples.

Dying samples with Nile Red can also overcome identification limitations of microscopy. This dye binds to polymeric materials facilitating rapid detection and quantification of microplastics, reducing the time required to analyse environmental samples. Washing of analysed particles with nitric acid digests any biogenic material in the dyed samples, ensuring the dye has not identified any products similar to microplastics, such as fatty substances or small fragments of debris.

Even though using microscopy to identify microplastics seems promising, extensive research is still required to develop a method of identification and characterisation that is efficient enough to detect all microplastics.

Pic: Shutterstock

Dr Rachel Sully is senior formulation scientist at SiSaf

Notes

¹ Mariano S, Tacconi S, Fidaleo M, Rossi M and Dini L (2021) Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques. Front. Toxicol. 3:636640. doi: 10.3389/ftox.2021.636640