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Differential staining lowers the false positive detection in a novel volumetric measurement technique of microplastics
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A novel volumetric microplastic detection method using Nile Red staining was refined with differential counterstaining to reduce false positives caused by non-plastic organic materials like lignin and chitin. The improved method was validated across soil, water, and food matrices, demonstrating better specificity for true microplastic detection.
A novel method for the volumetric detection of microplastics in various environmental (soil, water) and food (fish, meat) matrices was developed. The method is based on the Nile Red staining of microplastics while eliminating probable interference by other organic polymers such as lignin, chitin, cellulosic materials, and other organic substances using a mixture of three water-based dyes (Calcofluor White, Evans Blue, and 4,6-diamidino-2-phenylindole [DAPI]). The excitation/emission 'sweet spot' was determined for water based blue dyes to detect them in a single channel for effective elimination of probable contaminations. Detection of microplastic particles using the Nile Red method was validated by comparing with traditional detection of microplastics via Fourier transform infrared spectroscopy (FTIR). Volumetric measurements of the microplastics present in environmental samples were made possible using Z-stack confocal microscopy images backed by threshold-based 3D segmentation. Regularly shaped microplastic materials were used to validate the volumetric measurement method. The proposed volumetric determination method will be very useful for screening microplastics in diverse media and improving the prevailing method using FTIR.
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This study evaluated Nile Red fluorescent staining as a method for quantifying microplastics in environmental samples, comparing it to traditional identification techniques. The approach can help distinguish microplastics from organic particles more quickly and cost-effectively, supporting higher-throughput microplastic analysis in environmental monitoring programs.
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Researchers expanded the Nile Red staining method to quantify nanoplastics smaller than 1 µm, identifying fluorescent aggregates as a source of false positive counts and developing methodological corrections to overcome this challenge and improve the accuracy of nanoplastic detection in environmental samples.
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