Frequency‐Dependent Diffusion–Relaxation Distribution MRI: Scan–Rescan Reproducibility Ex Vivo and Caveats

Frequency-dependent diffusion-relaxation distribution MRI provides information beyond the traditional voxel-averaged metric that may better characterize the microstructural features of biological tissue. Frequency-dependent multidimensional ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>ω</mml:mi></mml:mrow> <mml:annotation>$$ \omega $$</mml:annotation></mml:semantics> </mml:math> MD) MRI reproducibility has been established in clinical settings, but has yet to be thoroughly evaluated under preclinical conditions, where superior hardware and modulated gradient waveforms enhance its performance. In this study, we investigate the reproducibility of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>ω</mml:mi></mml:mrow> <mml:annotation>$$ \omega $$</mml:annotation></mml:semantics> </mml:math> MD-MRI using a micro-imaging system to investigate ex vivo mouse brains. Notably, the estimated signal fractions of intra-voxel spectral components in the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>ω</mml:mi></mml:mrow> <mml:annotation>$$ \omega $$</mml:annotation></mml:semantics> </mml:math> MD-MRI distribution, corresponding to white and gray matter, along with the frequency-dependent parameters, demonstrated high reproducibility. We identified bias between scan and rescan in some of the metrics, which we attribute to the time gap between repeated scans pointing to a long-time progressive fixation effect. We compare our results with in vivo results from clinical scanners and show the reproducibility of diffusion frequency-dependent metrics to benefit from the improved gradient hardware on our preclinical setup. Our results inform future micro-imaging ex vivo studies of the reproducibility of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>ω</mml:mi></mml:mrow> <mml:annotation>$$ \omega $$</mml:annotation></mml:semantics> </mml:math> MD-MRI metrics and their dependence on fixation time.