## 3 Results and Discussion
### Experiments with Yeast Cells  
The spectra of yeast cells without spin probes were initially detected in different temperatures to define the baseline for the data obtained. The exemplary ESR spectrum of yeast cells is shown in Figure S5 (supplementary material) and is characterized by the absence of signals in the measuring range of TEMPO and TEMPOL. The subsequent studies concerned the mixture of the spin probes with yeast cells. The preliminary ESR measurements were carried out at 295 K. However, the possible location of the spin probes could not be determined based on the results obtained. The differentiation in ESR spectra was only observed for measurements carried out at 240 K, while the spectroscopic parameters were very similar for those recorded at 295 K (Figure 2). Different possible locations of the spin probes in yeast cell solution could be identified based on varied parameters of the ESR spectrum. Figure 3 demonstrates the schematic diagram of several different locations of spin probes in yeast cells and the corresponding ESR spectra of TEMPO and TEMPOL recorded at 240 K that reflect the possible presence of a spin probe in a given location. As yeast cells' incubation progresses over time, the structure of ESR spectra changes as a result of different environments. Thus, we can assume that the spin probes studied are mobile in cellular solution, and the corresponding spectra reflect subsequent steps of their movement into and within cells. ESR spectra should be different for spin probes outside cells, attached to the cell membrane, or inside a cell--probably near the mitochondria or between the outer and inner membranes where radicals (ROS) initially appear, originating, e.g., from the respiratory chain [2; 4; 5], as well as those inside cells undergoing intense disappearance. ESR spectra can also reflect the mixture of three different locations of the spin probe inside cells. It depends on the ratio of cells to spin-probe molecules, the rate of endocytosis, and the number of free radicals/ROS inside cells. The ESR spectra of TEMPO and TEMPOL reflect the different rates of penetration into cells but show an analogous relationship between the spectral shape and the possible location. A spin probe in a water environment has a single line in the ESR spectrum after freezing the samples to 240 K (Figure 3, spectrum 1). It is the effect of sample freezing, and thus decreasing the dynamics of the molecules and inhibiting rotation. However, the interactions between molecules are still manifested by a small spectrum width (1.24 mT). The single line is observed for both spin probes studied after mixing with yeast cells. A similar situation can be observed at room temperature when the concentration of the spin probe is very high [51]. The following possible spin-label location is much more easily determined for the samples containing TEMPOL. In contrast to the measurements at 295 K, the spin probe attached to the cell membrane has no such freedom of movement [52; 53; 54], and a wide triplet is observed (Figure 3, spectrum 2). Spectrum 2 was obtained for TEMPOL with yeast cells after 3 min of incubation. In contrast, the samples with TEMPO were characterized by a spectrum with different structures simultaneously (spectrum 3). Therefore, a lower penetration rate of TEMPOL into cells enables a more detailed analysis of spin probe location using the ESR method. A narrow triplet (Figure 3, spectrum 3) is attributed to a spin probe inside the cell characterized by high mobility. It is observed for TEMPO with yeast cells after only 3 min of incubation, while for TEMPOL after 2 h. Spectrum 4 in Figure 3 is characterized by a much lower concentration of spin probes. The differences observed result from the effect of yeast cell incubation time on spin-probesignal disappearance. Spectrum 3 was recorded earlier, while spectrum 4 after 1 h for samples with TEMPO. Additionally, the difference observed could also be a consequence of spin-probe penetration into cell organelles (e.g., mitochondria).