The glowing potential of Nile red for microplastics Identification: Science and mechanism of fluorescence staining

Quantification and identification of microplastics (MPs) (plastics < 5 mm) remain challenging due to the current laborious, inconsistent, and time-intensive measurement techniques. A promising time- and cost-effective alternative is staining with the fluorescent dye Nile red (NR). This review covers the wide range of NR staining methods, illumination conditions, and instrumentation for fluorescence-based detection and classification that have been developed thus far, highlighting the potential of NR fluorescence imaging to distinguish plastics. Despite notable advancements in NR staining techniques and conditions that have strengthened detection capabilities, there remains a need for further development and standardization of NR staining protocols, fluorescence imaging methods, and illumination instruments. We conduct a thorough assessment of both the advantages and limitations associated with diverse fluorescence imaging instruments, image segmentation, and classification techniques employed for detecting NR fluorescence and identifying polymer species. We also highlight critical considerations that should guide future research efforts to establish NR staining as a comprehensive, standalone method for environmental monitoring. They include investigating fluorescence behavior, especially intensity and Stokes shift, to understand the impact of solvent functional groups, plastic materials, additives and color pigments, weathering and application methods on NR sorption and fluorescence. Consideration of these factors will improve the ability to accurately identify polymer types based on their fluorescent behaviors, promoting widespread adoption of fluorescence imaging as a standalone method and enhancing cross-compatibility between NR studies.