Analyzing microplastics with Nile Red: Emerging trends, challenges, and prospects

The development and applications of effective analytical techniques for identification and quantification of microplastics in diverse spheres are increasing in the scientific arena. Nile Red (NR) staining has progressed as a low-cost, simple-to-use approach for analyzing the environmental impact of a wide spectrum of microplastics (e.g., ≥ 3 µm - ≤ 5 mm; polyethylene, polypropylene, and polyvinyl chloride etc.). Given the recent surge of research into this methodology, it is critical to examine the findings and present future directions. Herein, we review accomplishments to date of the current protocols describing the sample preparation, staining and fluorescence conditions, contamination measures, and data analysis based on 56 field observations focusing on microplastic pollution and NR staining technique. Additionally, we discuss the challenges in current analyses towards standardization and recommendations related to it. Finally, we conclude that, despite methodological discrepancies, the NR method has emerged as a viable standalone substitute for visual identification; yet not all that fluoresce with NR are microplastics, which necessitates extensive sample preparation or additional spectroscopy techniques for chemical analysis to validate the results. This article informs the reader about how the NR technique is advancing microplastic research and identifies current needs for future advancements.