• Inhomogeneous magnetization transfer (ihMT) was validated as a myelin sensitive imaging technique against fluorescence microscopy • ihMT signal was strongly and significantly correlated with myelin-related plp-GFP (proteolipid-Green Fluorescence Protein) signal • Short dipolar relaxation time (T1D) filtering is an efficient way to reduce non-myelin contribution in ihMT signal • MT signal was more weakly correlated with plp-GFP signal and had a much larger nonmyelin contribution • ihMT contrast can be varied with pulse timing, leading to different signal properties in terms of sensitivity and specificity for myelin content Data availability statement Data (Bruker format file) will be shared by request from any qualified investigator.

T 1D , the relaxation time of dipolar order, is sensitive to slow motional processes. Thus T 1D is a probe for membrane dynamics and organization that could be used to characterize myelin, the lipid-rich membrane of axonal fibers. A mono-component T 1D model associated with a modified ihMT sequence was previously proposed for in vivo evaluation of T 1D with MRI. However, experiments have suggested that myelinated tissues exhibit multiple T 1D components probably due to a heterogeneous molecular mobility. A bi-component T 1D model is proposed and implemented. ihMT images of ex-vivo, fixed rat spinal cord were acquired with multiple frequency alternation rate. Fits to data yielded two T 1D s of about 500 µs and 10 ms. The proposed model seems to further explore the complexity of myelin organization compared to the previously reported mono-component T 1D model.