Highly efficient Nile red staining for the rapid quantification of microplastic number concentrations using flow cytometry

Fluorescent staining of microplastics (MPs) with Nile red (NR) improves the sensitivity of optical techniques, including microscopy and flow cytometry (FCM), and is commonly employed to quantify MP number concentrations. However, staining efficiency varies depending on the polymer type and surface characteristics, often resulting in insufficient fluorescence intensity. This study presents an improved protocol using a swelling-mediated NR staining method, which enables NR to be encapsulated within MPs rather than merely adsorbed on the surface, as in conventional methods. This technique yields strongly fluorescent MPs, improving detection sensitivity and allowing identification of MPs smaller than a few micrometers. Using this method in combination with FCM, we successfully quantified MP number concentration for particles with diameters less than 10 µm. The pretreatment and staining procedures were optimized using polystyrene particle suspensions (2, 5, and 10 µm) with known number concentrations. The staining efficiencies were close to 1. Recovery tests were also carried out using simulated environmental samples with tap water as the matrix. After correcting for the FCM's counting efficiency, the recovery rates were 0.993 ± 0.038 (2 µm), 0.988 ± 0.029 (5 µm), and 0.846 ± 0.100 (10 µm). Differences in recovery rates were attributed to instrument-specific counting efficiency, with no evidence of systematic uncertainty associated with the staining process. Our findings indicate the effectiveness of the swelling-mediated NR staining technique for quantifying MPs and highlight its potential as a rapid and reliable approach for analyzing MPs in environmental samples when combined with high-throughput FCM.