Easy and accessible way to calibrate a fluorescence microscope and to create a microplastic identification key.

We present here a technique for setting up detection limits on any fluorescent microscope in conjunction with the fluorophore Nile Red for microplastic identification. Our method also describes a rigorous morphology-specific identification key for microplastics to reduce subjectivity between researchers. The detection limits were established for nine common polymer types and five natural substrates which could result in false-positive signals when using Nile Red for microplastic identification. This method was then applied to real freshwater samples and identified particles were validated with micro-FTIR or Raman spectroscopy. This approach may reduce subjectivity in microplastic identification and counting and enhances transparency, repeatability and harmonization within microplastic research community.•Instructions for calibration of detection limits for microplastics on fluorescence microscope systems described•Microplastic identification key developed and tested to reduce false positive detection•Lower subjectivity for microplastic identification obtained using the detection limits & identification key.