Systematic optimization of Nile Red staining parameters for the reliable detection of microplastics ≤ 2 µm

Detection of small microplastics remains analytically challenging despite their growing environmental and biological relevance. Nile Red staining offers an affordable alternative to state-of-the-art techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy. However, it has been mainly used for larger particles, typically > 20 µm. This study presents an optimized filtration and Nile Red (NR) staining protocol for the reliable detection of microplastics smaller than 5 µm, with particular emphasis on particles ≤ 2 µm. Filtration conditions and staining parameters, including NR concentration and incubation time, were systematically optimized to enhance detection efficiency. Under these conditions, polystyrene (1 µm and 2 µm) and polyethylene (2 µm) particles exhibited strong and consistent signals, while inorganic controls showed no staining, confirming dye specificity. Formalin fixation, a treatment often applied to conserve environmental samples for later analyses, further improved particle brightness and signal-to-noise ratio without compromising selectivity, as no fluorescence was observed in fixed biological material. Our simple, cost-effective protocol can be applied in labs with lower resources and also for initial screening of particles in natural samples for further analyses.