# Microplastics' Detection in Honey: Development of Protocols in a Simulation
## 3 Results and Discussion
### Method 2: PET MPs Detection in Honey through Optical Observation of Fluorescence
#### 3.2.2 Enhanced Optical and Fluorescence Microscopy with DANS Dye under UV  
Radiation and Multispectral Imaging  
In these experiments, DANS dye was chosen to improve the detection efficiency of MPs on CA filters under UV radiation. PET MPs were dyed inside the honey environment. MPs presented a significant fluorescence, even though samples were thoroughly washed using pentane. For the identification of MPs, Raman measurements were conducted as mentioned in Section 2.6.1 for Protocol 3. Several dimensions of MPs were detected: 3 \(\upmu\)m, 5 \(\upmu\)m, 10 \(\upmu\)m, 18 \(\upmu\)m, 20 \(\upmu\)m, 35 \(\upmu\)m, and 40 \(\upmu\)m. Figure 6 indicates the Raman spectral signatures with the fluorescence due to the DANS dye absorbed from MPs.  
PET MPs can also be dyed with DANS by passing the dye through the syringe filter. Afterward, the syringe filter was left at room temperature (RT) to dry for 24 h, before breaking the filter cup to collect the filter. Alternatively, one can break the filter cup and spread the dye on the filter surface afterward, but it was preferable to pass the dye through the syringe filter before breaking the filter cup because the dye spreads more homogeneously on the CA filter. Optical fluorescence measurements in combination with Raman measurements were conducted. Also, DANS dye does not interfere with Raman signals of plastics. An important observation was also that honey solid particles do not present this specific \"glowing crown\". Honey solid particles have a red and bright yellow fluorescence, while the clean CA filter appears light pink under UV radiation. It was assumed that it could be possibly used for the identification of honey MPs in real samples. It was concluded that the less time-consuming way to dye PET MPs was to pass the dye  
Figure 5: (**a**) PET MPs with honey solids on CA filter; (**b**) PET MPs with honey solids on the microscopic metallic slide; (**c**) The Raman signals of the five detected PET MPs from Figure 5a,b and Figure 4a–c compared with PET powder Raman signal.  
solution through the syringe filter before breaking the filter cup. Still, the PET MPs were more homogeneously dyed when the dye solution was mixed with a honey sample with double quantity based on the [23] protocol. In Figure 7a,b dyed PET MPs are presented under UV, observed with a Raman microscope, whereas Figure 7a refers to dyed PET MPs under UV on a CA filter from the first experiment, and in Figure 7b, to the second experiment. Dyed PET MPs are distinguished by the \"glowing crown\" as mentioned previously. In Figure 7c1,c2 dyed PET MPs are observed under UV on a CA filter in honey. As was mentioned before, the CA filter with MPs was dyed with DANS dye in two different ways and allowed to dry at room temperature. In these pictures, solid particles of honey are observed, which have red and yellow colors under UV radiation. Additionally, in Figure 7d pure honey with DANS under UV is presented on the CA filter. Here, solid particles of honey are observed, which have red and bright yellow colors under UV radiation. A pure filter with DANS is presented in Figure 7e, where there are not any dyed particles. Finally, in Figure 7f, dyed MPs under UV are presented, in comparison with Figure 7g, where the same area as the picture area is presented with monochromatic imaging through a multispectral device (Xpecam X02, XpectralTEK, Braga, Portugal) at 625 nm wavelength. Multispectral imaging was used to enhance the contrast between the dyed PET MP particles and the background. The resulting images did not show a significant enhancement, with the best contrast appearing at 625 nm.  
Figure 6: (**a**) Picture with MPs from first experiment with dyed MPs into the honey; (**b**) Raman plot of MPs detected in picture (**a**); (**c**) Picture with MPs from second experiment with dyed MPs in honey, before dyed MPs mixed with it; (**d**) Raman plot of MPs detected in picture (**c**).