## 3 Results and Discussion
### Experiments with Yeast Cells
The proof that we realistically monitored the process of TEMPO entering the cell was described in our previous work [27], presenting an experiment with sample filtering. When we filtered the incubated sample in which there was endocytosis (a narrow triplet at 240 K), we did not see any signal in the filtrate. However, the ESR signal was visible in the material left on the filter. It confirms that the signal observed by us at 240 K results from changes in the sample during incubation (endocytosis) and not of low temperature. If we kept the sample with the cells and the spin probe at the temperature of 273 K (this is the temperature at which endocytosis does not occur), after filtering the sample, we recorded a single line at the temperature of 240 K, such as in Figure 3, spectrum 1, confirming the presence of TEMPO in the solution and not attached whether present in the cell. The study on the spin probes' endocytosis process will be continued. The location of spin probes was also analyzed by analogous experiments carried out in the presence of a fluorescent spin-probe reagent (TEMPO-Fluorescein). TEMPO-Fluorescein was added to the yeast cell suspension, and the process was monitored by the ESR method and confocal microscope (Figure 4). As can be seen in the pictures taken under the confocal microscope, the fluorescence from TEMPO-Fluorescein located in the cell membranes is visible as a delicate envelope around the yeast cells. If we compare Figure 4a,b, it can be seen that this fluorescence comes from only a few cells. The reference sample of TEMPO-Fluorescein without cells measured at 240 K was characterized by the single line in the ESR spectrum (data not shown), while the one combined with cell suspension was by a wide triplet. Unfortunately, the signal intensity and the spectrum structure did not change during incubation, even after 24 h. It can be assumed that the modified TEMPO-Fluorescein molecules are only attached to a cell membrane and cannot penetrate cells. The endocytosis process is prolonged or does not occur (Figure 4). The same signal intensity indicates no redox reaction in the system. In contrast, the same spectrum  
Figure 3: Schematic representation of possible spin probe location based on ESR spectrum shape of TEMPO or TEMPOL with yeast cells recorded at 240 K: 1—spin probe outside cells, 2—spin probe attached to a cell, 3—spin probe inside cells, 4—spin probe inside cells (probably in cellular mitochondria). The figure was created using Servier Medical Art templates, licensed under a Creative Commons Attribution 3.0 Unported License; [[https://smart.servier.com](https://smart.servier.com)]([https://smart.servier.com](https://smart.servier.com)) (accessed on 16 December 2019). structure (wide triplet) suggests that the environment of the molecules studied at different incubation times remains unchanged. This was confirmed by the water-organic liquids boundary-crossing experiment for TEMPO and TEMPO-Fluorescein. The partition coefficient (P) is the term used to define the ratio of the concentrations of substances, TEMPO or TEMPO-Fluorescein spin probe, in two immiscible solvents at equilibrium. Therefore, this ratio is a comparison of the solubility of the solute in these two liquids. The study of TEMPO or TEMPO-Fluorescein spin probes' diffusion through the water-ethyl acetate (w/ea) or water-hexane (w/h) phase boundaries was carried out in a glass vessel. A total of 150 \(\upmu\)L of an aqueous solution of spin probes containing 0.06 mg of TEMPO or 0.03 mg of TEMPO-Fluorescein and 150 \(\upmu\)L of organic solvent was placed. The contact area of the water-organic solvent phases in the measuring vessel was 154 mm\({}^{2}\). The concentration of spin probes in both phases was measured by the ESR method by collecting samples of solutions to capillary, with a capacity of 16 \(\upmu\)L, after the equilibrium was established (2.5 h).