The development and application of four-dimensional spectral-spatial electron paramagnetic resonance imaging (EPRI) techniques for unambiguous determination of spectral shape and spin distribution of paramagnetic samples are described. Strategies for optimizing acquisition and computation times, image resolution, and data presentation are described. The feasibility of studying small linewidth differences of approximately 0.1 G in samples of up to 25 mm in size was tested by computer simulations and by measurements on phantoms containing 0.5 mM nitroxide spin label. One thousand projections were used to reconstruct 32 × 32 × 32 × 32 pixel images. Similar 4D imaging experiments were performed on an isolated rat heart infused with a suspension of glucose char. The 4D image of the rat heart clearly showed the three-dimensional spatial structure of the heart and the spectral shape at each spatial point. Thus, we have demonstrated, for the first time, that 4D spectral-spatial EPRI could be performed on lossy biological samples or tissues at L-band frequencies, enabling the mapping of spectral information over the entire three-dimensional spatial structure of the object.
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