## 3 Results and Discussion  
The studies concerning the structure and dynamics of biomolecules in cellular solution or inside cells are crucial for understanding molecular-level processes. The biological systems and processes inside cells were described using different experimental methods, i.e., mass spectrometry [44; 45], fluorescence spectroscopy [46; 47], or magnetic resonance spectroscopy [48; 49]. However, the application of Site-Directed Spin Labeling (SDSL) coupled with EPR spectroscopy was found to be suitable for describing biological macromolecules (proteins and nucleic acids) in cellular conditions [50]. The use of spin probes as structural, redox or oxygen probes in the SDSL-EPR method enables detailed studies of interactions in biological systems. On the other hand, the technique requires further improvement to develop a standard procedure for various biomolecules. The present studies demonstrate the effect of cumulative interaction of various components in biological systems reflected by ESR spectra changes.  
Biological studies using spin probes and ESR are generally carried out at room temperature (RT). It should guarantee significant changes in signal intensities and dynamics. However, in the present studies, the differentiation of ESR signal parameters in such conditions was negligible, which could be explained by significant dynamics in the system studied that exclude observation of substantial changes. The interaction anisotropy of the spin probes with a diverse environment is averaged, and the differences in spectroscopic parameter values are insignificant or within the measurement accuracy. The ESR spectra of TEMPO and TEMPOL with yeast cells recorded at RT are shown in Figure 2a,c, respectively. The spectra differ only in a superhyperfine structure that did not change significantly during cell incubation. Therefore, ESR measurements were carried out at room and lower temperatures to observe the differences in dynamics, hyperfine structure, and the anisotropy of ESR spectra. Based on many years of ESR research experience using spin probes and biological samples and previous results, a temperature of 240 K was chosen as an optimal for differentiating the properties and dynamics of the spin probes during incubation [26; 27]. We noticed that at this temperature (240 K), the best differentiation of the signal and its parameters is observed (Figure 2b,d). After sample freezing, the dynamics are reduced significantly, and the differentiation can be observed.  
The spin probes studied are very stable in aqueous solutions. However, combining their solution with cells results in rapid recombination at 310 K (incubation temperature). The effect of several factors, including spin probe concentration, presence of oxygen and  
Figure 2: ESR spectra of TEMPO (**a,b**) and TEMPOL (**c,d**) spin probes with yeast cells recorded at 295 K (**a,c**) and 240 K (**b,d**).  
vitamin C, and the type of cells, affecting the time of a selected spin-probe signal disappearance was successively analyzed. The nitroxide radicals studied (TEMPO and TEMPOOL) differ in structure, as the latter has a hydroxyl group, which can affect the compounds' biological activity. According to the literature reports [29], TEMPO with lipophilic nature is more cytotoxic than hydrophilic TEMPOL, as it tends to accumulate in the cell membrane. Therefore, the nitroxide radicals studied could be responsible for activating different cellular signaling pathways. It can be assumed that their other behavior in biological systems should be manifested in ESR spectra.