# Accumulation of Spherical Microplastics in Earthworms Tissues-Mapping Using Raman Microscopy
## 2 Materials and Methods
### Study of Microplastics in Earthworm Tissues  
Raman spectroscopy was performed on WITec Alpha 300R and WITec Alpha 300 RSA+ spectrometers. Both spectrometers are equipped with a confocal microscope and a TrueSurface attachment. In order to detect the analyzed microplastics in earthworm tissues, three excitation lines were tested: 532, 633, and 785 nm. Due to the strong fluorescence of microplastics during measurement using the 532 nm line, it was not possible to record Raman spectra. In the case of the 785 nm line, a spectrum was obtained for GreenPEM microplastics, while for VioletPEM, mainly fluorescence was observed, which was difficult to distinguish from the fluorescence of the own tissue. Therefore, the measurements were performed using a laser excited at a wavelength of 633 nm.  
Using this laser line, it was possible to obtain a spectrum for GreenPEM microplastics. For the VioletPEM microplastics, strong fluorescence with a characteristic spectral profile was observed, allowing the spectra to be distinguished from the intrinsic fluorescence of the examined tissues with high efficiency. In the first stage, single spectra of microplastic samples were made with the following parameters: accumulation number of 10, single spectrum accumulation time of 0.5 s, objective lens with 20\(\times\) magnification. In the case of GreenPEM microplastics, the radiation power was 12 mW, while for VioletPEM, it was set to 1 mW. Raman mapping involved the sequential collection of individual spectra from a specific surface. Each time, the spectrum was measured over the entire spectral range, i.e., 0-2800 cm\({}^{-1}\). Images were obtained using a 10\(\times\) (NA 0.25) or 20\(\times\) (NA 0.5) objective.  
Measurements were carried out with the following settings:  
Figure 1: (**A,B**) Emission and excitation peak of the polyethylene used.  
(a) Mapping large tissue fragments: sampling density of 40-10 \(\upmu\)m, map size depending on the tissue size, spectrum accumulation time of 0.1 s, radiation power on the sample of 9-12 mW.  
(b) Mapping of smaller tissue fragments: sampling density of 5 \(\upmu\)m, map size of 500 \(\times\) 500 \(\upmu\)m, spectrum accumulation time of 0.1 s, radiation power of 9-12 mW. The sampling density was experimentally adjusted to the size of the microplastics in the tissues. Raman images were prepared based on the True Component Analysis (TCA). This analysis makes it possible to identify different components in the sample based on Raman spectra and create an average spectrum for them. The result also includes images showing the distribution of the accumulation of components identified in the sample. The TCA image is constructed in such a way that intense yellow pixels indicate high spectral intensity for the identified component. TCA analysis made it possible to isolate classes containing spectra corresponding to the spectral profile of microplastic standards (control samples).  
The GreenPEM spectrum has a set of characteristic bands at 687, 1215, 1296, 1446, and 1538 cm\({}^{-1}\) (Figure 2A). In turn, for the VioletPEM microplastics, very strong but characteristic fluorescence is observed, present even at low laser powers (1 mW) (Figure 2B). It is important that the fluorescence spectrum profile is different and more intense than the fluorescence of the tissue itself, making it possible to distinguish this microplastic from the tissue.