Pulsed EPR imaging of nitroxides in mice

Nitroxides, unlike trityl radicals, have shorter T₂s which until now were not detectable in vivo by a time-domain pulsed Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz since their phase memory times were shorter than the spectrometer recovery times. In the current version of the time-domain EPR spectrometer with improved spectrometer recovery times, the feasibility of detecting signals from nitroxide radicals was tested. Among the nitroxides evaluated, deuterated ¹⁵N-Tempone (¹⁵N-PDT) was found to have the longest T₂. The signal intensity profile as a function of concentration of these agents was evaluated and a biphasic behavior was observed; beyond a nitroxide concentration of 1.5 mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with ¹⁵N-PDT in solutions equilibrated with 0%, 5%, 10%, and 21% oxygen using the single point imaging (SPI) modality in EPR. The image intensity in these tubes was found to depend on the oxygen concentration which in turn influences the T₂ of ¹⁵N-PDT. In vivo experiments were demonstrated with ¹⁵N-PDT in anesthetized mice where the distribution and metabolism of ¹⁵N-PDT could be monitored. This study, for the first time shows the capability to image a cell-permeable nitroxide in mice using pulsed EPR in the SPI modality.