## 6 Methodology for MP Analysis
### Analytical Methods for Identification, Quantification and Characterization of MPs
#### 6.3.1 Microscopic Methods  
Light microscopy can be applied to analyze the physical properties of MPs, such as the color, shape, size, surface structure and degree of corrosion and weathering [14; 64; 105]. For morphological characterization and counting of MPs, one can use a stereoscopic (dissection) microscope for particles larger than 50 \(\upmu\)m [41; 64] or a classic optical microscope with 10-50\(\times\) magnification [54]. These devices can be connected to a computer and combined with image analysis programs [54], e.g., Image J [41]. Visual detection is suitable only for clean matrices [76] and if the MP particles are not transparent [54]. The disadvantage of the method is the possibility of misidentification of MP particles due to non-recognition or confusion with other materials (e.g., coal ash, paint fragments, natural fibers: cellulose, keratin and viscose) [14; 58; 54; 54].  
Electron microscopes can compensate for the shortcomings of optical microscopes because they reach higher magnification and resolution, thus providing a clearer structural image of the MP surface [64]. For analyzing the surface morphology of MPs, a scanning electron microscope (SEM) is mainly used. An SEM can analyze particles up to 1 nm in size [105] and provides data on size, shape, surface structure, wear, degradation and microbiological colonization [52]. The preparation of samples for SEM microscopy requires additional pre-treatment of coating MP particles with certain chemicals [52; 57], so it is not suitable for analyzing the surface structure and color [14].  
A polarized light microscope (PLM) can be used to analyze some types of MPs whose crystal structure allows for light to pass through, but the method is not suitable for opaque and thicker MPs [64]. The use of hydrophobic fluorescent dyes for MP staining [105], such as the Nile Red reagent (NR staining method) are shown to be effective in the analysis of sand samples [53]. The stained samples are then analyzed with a fluorescence microscope or a confocal laser scanning microscope. Staining with fluorescent dyes can give false positive results because the bioorganic material contained in the sample is simultaneously stained, and the environmental samples can have fluorescent properties by themselves [105].  
The type of polymer cannot be determined by microscopic techniques because they do not provide information on the composition of MP particles [104]. To obtain reliable results, microscopy can only be used as a preliminary technique in combination with other methods. For a simultaneous analysis of the surface structure and elemental chemical composition of MP particles, a combination of an SEM with energy dispersive X-ray spectroscopy (SEM-EDX) can be used [64; 105]. A prerequisite for this analysis is the conductivity of the samples. MPs are not conductive, so they require a complex pre-treatment (e.g., gold plating); this technique is currently used only for the analysis of certain types of MPs and is not applicable for environmental samples [105]. Combinations of microscopy with different techniques is recommended, and the most commonly used combinations are the ones with spectroscopic techniques (\(\upmu\)-FTIR and \(\upmu\)-Raman) [41].