The strengths of Magnetic Particle Imaging (MPI) lay in its sensitivity, quantitative nature, and lack of signal attenuation for Superparamagnetic Iron Oxide Nanoparticles (SPION). These advantages make MPI a powerful tool for the non-invasive monitoring of tracer behaviour over time. With more MPI studies emerging, a standardized method for determining the boundaries of a region of interest (ROI) and iron quantification is crucial. The current approaches are inconsistent, making it challenging to compare studies, hindering MPI progression. Here we showcase three different ROI selection methods for the quantification of iron in vivo and ex vivo. Healthy mice were intravenously administered a long circulating tracer, never before applied in MPI, and the ROI methods were tested for their ability to accurately quantify the total signal present, in addition to the accumulation of the tracer in individual organs. We discuss how the quantified iron amount can be vastly altered based on the choice of ROI, the importance of the standard curve and the challenges associated with each method. Lastly, the user variability and accuracy of each method was compared by 3 independent users to ensure their consistency and lack of bias.
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