A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

Abstract

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Figure 1. 1 g of marine sediment (SPI 6) spiked with microplastics of six different polymer types, dyed with Nile Red (1000 μg mL⁻¹, 30 minutes), then filtered onto a 47 mm diameter membrane filter.

Photograph taken with a blue light (Crime Lite: 450–510 nm) and orange filter (529 nm).

Figure 2. Fluorescent index, represented by (R+G)/R, plotted against published static contact angle values (a measure of the surface polarity).

The actual images are inset to show the clear colour variations.

Figure 3. Filter images from processed sediment sample number 805.

(A) white light, showing a variety of extracted debris; (B) Autostitch reconstruction of the 54 tiled images taken using a blue light and orange filter, (C) expansion showing three bright spots of fluorescently-tagged microplastics and (D) close-up of one larger particle, approximately 100 μm across. A number of bright spots much smaller than this are also clearly visible in image (C).

Figure 4. Part of a filter image from sample 805, reconstructed from individual tiles, showing fluorescent particles and, superimposed, the IR spectra obtained by picking the three larger particles and transferring them to an Anopore filter.

This allowed them to be identified as common microplastics. Note also, the many additional small bright particles (25 have been ringed for clarity), which were too small to transfer reliably.

Figure 5

Image of the filter in white light showing (A) the scribed area; (B) expansion of the scribed area under blue light, photographed through an orange filter, reconstructed from tiled images showing the bright fluorescent objects identified, (C) tiled white-light image from the IR microscope overlaid with a C-H filtered IR spectral map to highlight organic material and below, IR spectra from the 5 locations ringed and numbered in panel (B).