What have we known so far for fluorescence staining and quantification of microplastics: A tutorial review

Understanding the fate and toxicity of microplastics (MPs, < 5 mm plastic particles) is limited by quantification methods. This paper summarizes the methods in use and presents new ones. First, sampling and pretreatment processes of MPs, including sample collection, digestion, density separation, and quality control are reviewed. Then the promising and convenient staining procedures and quantification methods for MPs using fluorescence dyes are reviewed. The factors that influence the staining of MPs, including their physicochemical properties, are summarized to provide an optimal operation procedure. In general, the digestion step is crucial to eliminate natural organic matter (NOM) to avoid interference in quantification. Chloroform was reported to be the most appropriate solvent, and 10-20 mu g/mL are recommended as optimal dye concentrations. In addition, a heating and cooling procedure is recommended to maintain the fluorescence intensity of MPs for two months. After staining, a fluorescence microscope is usually used to characterize the morphology, mass, or number of MPs, but compositional analysis cannot be determined with it. These fluorescence staining methods have been implemented to study MP abundance, transport, and toxicity and have been combined with other chemical characterization techniques, such as Fourier transform infrared spectroscopy and Raman spectroscopy. More studies are needed to focus on the synthesis of novel dyes to avoid NOM's interference. They need to be combined with other spectroscopic techniques to characterize plastic composition and to develop image-analysis methods. The stability of stained MPs needs to be improved.