Combining Low Cost Imaging and Fluorescence Spectroscopy to Assess a Nile Red Counterstain for Differentiating Microplastic and Organic Particles

Microplastics (MPs) are small fragments of plastic debris that have become pervasive in the natural environment due to their lightweight, durable nature and diminutive size. The fluorescent dye Nile Red has become increasingly popular for staining and detecting microplastics using the naked eye or visual imaging. However, microplastic detection using Nile Red alone is susceptible to false positives caused by stained or autofluorescent organic materials. Counterstaining, which introduces a fluorescent stain that binds preferentially to organic materials, is an emerging method to reduce false positives when tagging plastics with Nile Red. However, few studies characterize the fluorescence emission spectra of particles stained with Nile Red and a counterstain. To assess the viability of a counterstain for altering the spectra of organic materials that may otherwise be mistaken for plastics, fluorescence spectra were gathered for organic materials stained with Nile Red and a counterstain Calcofluor White and Evans Blue for the first time. The particles were then imaged alongside plastics stained with a mixture of Nile Red and the counterstain to ascertain if organic materials could be visually differentiated based on the counterstain. The presented methods contribute to developing accessible microplas-tic detection technologies,