Strategies towards standardizing calibration methods for magnetic particle imaging (MPI) signal quantification: solution <i>vs.</i> cellular environments

Magnetic Particle Imaging (MPI) is a powerful technique for non-invasive imaging and iron quantification using superparamagnetic iron oxide nanoparticles (SPIONs), with applications ranging from <i>in vivo</i> cell tracking to tracer distribution and biodistribution studies. As the MPI community continues to grow and diversify, there is an increasing recognition of the need for standardized approaches in signal quantification to ensure reproducibility, comparability, and reliable interpretation of results across studies. A key area where standardization is particularly needed is in the construction of calibration curves for quantitative MPI. In this study, we demonstrate that calibration curves derived from SPIONs in solution differ markedly from those obtained in cellular environments. We therefore propose calibrating MPI signal against the number of labelled cells, a strategy that accounts for altered SPION behaviours in the cellular environment and enables more accurate estimation of intracellular iron content. Another critical but often overlooked factor in MPI quantification is the influence of SPION concentration and spatial distribution within the sample. We show that even modest variations in concentration can significantly affect MPI signal intensity, challenging the commonly assumed linear relationship between signal and iron content. Our findings reveal that variations in concentration can introduce nonlinearities in signal response, thereby altering calibration curves and impacting the accuracy and reproducibility of MPI-based quantification. By systematically examining the effects of environmental context and SPION concentration, our study provides a framework for biologically relevant MPI calibration strategies and supports the development of more standardized, reproducible quantification protocols.