## 3 Results and Discussion
### The Influence of Oxygen in the Atmosphere and Vitamin C on Spin Probes in Cell Culture  
A reduced amount of oxygen can influence the formation of free radicals in a breathing cycle and the dynamics of endocytosis. Therefore, the measurements were performed for incubation in a nitrogen and oxygen atmosphere (Figure 7). Yeast cells are adapted to growth under aerobic and anaerobic conditions. The anaerobic atmosphere does not affect the reproduction process, but the number of reactive oxygen species and intracellular antioxidants in cells is significantly reduced [56]. The disappearance of spin probes is slower in the nitrogen atmosphere. It could be explained by the deactivation of aerobic respiration and thus lower production of radicals by mitochondria into the cytoplasm. The difference is particularly significant for TEMPOOL molecules with slower endocytosis than TEMPO. The presence of strong antioxidants can also influence endocytosis. Therefore, vitamin C was applied as a model-reducing agent and added to TEMPO solutions (Figure 9). The effect of vitamin C presence was studied for solutions of three different compositions: TEMPO with vit. C, TEMPO with yeast cells, as well as TEMPO with vit. C and yeast cells. In the absence of yeast cells, TEMPO recombination occurs as an effect of the redox process (Figure 10), while the signal fades much more slowly in the presence of yeast cells. Figure 8: The schematic representation of TEMPO (R: -H) and TEMPOOL (R: -OH) reduction to stable hydroxylines. Figure 7: Changes of TEMPO (**a**) and TEMPOOL (**b**) concentration in solutions incubated in nitrogen (\(\blacktriangle\)) and oxygen (\(\blacksquare\)) atmosphere. According to our results, endocytosis of vitamin C is much slower than that of TEMPO. Therefore, its participation in free radicals scavenging inside cells is invisible in the presence of TEMPO. Hence, applying TEMPO as a free radicals scavenger should be more effective than vitamin C. Additionally, the analogous experiments were applied to the samples with  
Figure 10: The influence of vitamin C on TEMPO concentration: yeast cells with TEMPO and vitamin C (\(\blacksquare\)), TEMPO with vitamin C (\(\bullet\)), and yeast cells with TEMPO (\(\blacktriangle\)). Figure 9: ESR spectrum of TEMPO spin probe with yeast cells in the presence of vitamin C recorded at 240 K (incubation time 10 min). TEMPOL. However, this spin probe penetrates much more slowly in cellular solution, and no significant changes were observed using the same incubation time range. The experimental data from Figure 10 were further used to create a mathematical model for describing individual endocytosis steps. The differences in signal intensity of TEMPO penetration and disappearance during incubation time can be estimated using Equation (2):  
\[\text{I(t)}=\text{Aout(t)}+\text{Am(t)}+\text{Ain(t)} \tag{2}\]  
where:  
\[\text{Aout(t)}=\text{C}\times\exp(-(\text{t}-1)/\tau 1) \tag{3}\]  
\[\text{Am(t)}=\text{Aout(t)}-(\text{C}\text{C}\times\exp(-(\text{t}-1)/\tau 1)) \tag{4}\]  
\[\text{Ain(t)}=\text{Am(t)}-\text{K1}\times\text{Am(t)}+\text{K1}\times\text{ Am(t)}\times\exp(-(\text{t}-1)/\tau 2) \tag{5}\]  
The I(t) function describes changes in TEMPO concentration over time measured in a suspension of cells with the spin probe. I(t) is the integral intensity of the ESR spectrum, and its value is directly proportional to the concentration of TEMPO particles in the cell suspension. Aout(t) describes the number of spin-probe molecules in suspension not associated with the cells. Am(t) is the number of TEMPO molecules attached to the cells, while Aim(t) is the number that entered cells during endocytosis and is recombined with free radicals inside the cells. The amounts of spin probes Aout(t), Am(t), and Ain(t) were calculated using Equations (3)-(5), respectively. The parameter C is the total amount of TEMPO molecules in a cell suspension. It is normalized to 100 a.u. (Figure 11). In most experiments, its absolute value was about 3 \(\times\) 10 \({}^{3}\) mol/L.