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EPR spectral sensitivity of the three biradicals of imidazoline and imidazolidine types to the local concentration of protons (pH) in aqueous solutions has been studied. Significant increase in g-factor (Ag 0.0002) and decrease in hyperfine interaction constant (AaN = 0.8-1.2 G) upon the protonation of atom N-3 of the radical heterocycle were observed. The pH dependence of a N and gfactor is described by conventional titration curve with one or two pK values. The latter case was explained by nonindependent protonation of two monoradical fragments closely located in the biradical structure. EPR spectra of protonated and deprotonated forms of the biradicals have significantly different intensities of "biradical" components, Ib, reflecting changes in spin exchange interaction. It has been found that activation energy of spin exchange, Ea, estimated from temperature dependence of Ib in glycerol/water solution is lower for the biradical conformation with larger I b . This is in agreement with main contribution of direct collisions of monoradical fragments in spin exchange interaction so larger value of Ib corresponds to the conformation with less spatial restrictions for the motion of monoradical fragments (lower Ea). The potential use of the pH-sensitive biradicals as spin labels is discussed.

The fluorescence photobleaching recovery method with pulse-position modulation (PPM) of bleaching/probing irradiation is presented. This approach is modification of previously announced photobleaching recovery under decaying photobleaching (FRDP) approach, which employs the amplitude control of irradiation intensity. Underlying idea was to solve the problem of setup nonlinearity by methodical modification that was successfully done. The irradiation intensity is series of equal bleach-probe pulses with increasing distance. The more sophisticated series was proposed as a convolution of subseries, which one characterizing its own time scale. It allows to detect the fluorescence kinetics in the wide range of time and of different nature simultaneously. The method was applied on model systems as FITC-HSA in the water/glycerol mixture and FITC-DMPE in the POPC multibilayers. The method allowed to detect and investigate diffusion processes, presence immobile fraction, effect of limited area of liposomes and reversible fluorescence kinetics.

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