The use of pH-sensitive nitroxides, in conjunction with low-frequency EPR, offers a unique opportunity for non-invasive assessment of pH values (in the range 0 to 14) in living animals. In the present study, we have investigated the potential use of pH-sensitive nitroxide free radicals in conjunction with EPR imaging techniques at low and very low frequencies (280 MHz-2.1 GHz). In particular, we have measured the hyperfine splitting (hfs) of a pH-sensitive probe at three different EPR frequencies: 280 MHz, 1.1 GHz and 2.1 GHz. We have also developed EPR imaging experiments with phantoms simulating in vivo conditions, using pH-sensitive probes at 280 MHz (spatial-spatial) and 1.1 GHz (spectral-spatial). Finally, we discuss the actual sensitivity/resolution limits of the EPR imaging techniques at low frequencies. Practical applications of this method in the biomedical field are suggested for the continuous and non-invasive localization of pH in vivo.

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