Researchers and clinicians interested in non-invasive characterization of tissue microstructureThe aggregate myelin g-ratio (see Fig. 1) is a function of the myelin volume fraction (MVF) and the fiber volume fraction (FVF) ($$$g = \sqrt{1 - \frac{MVF}{FVF}}$$$)^{1, 2}. While this relationship holds in theory³, obtaining precise and accurate MRI measures of the MVF and FVF remains a challenge. In this work, we focus on three magnetization transfer (MT) derived MVF metrics and explore how improper calibration of the MVF estimates propagates to the aggregate g-ratio. We performed magnetization transfer and diffusion imaging (2mm isotropic resolution) on one healthy subject and one multiple sclerosis (MS) patient. For the MS patient, T2W and PDW images were acquired for lesion segmentation. For each subject we calculated three MRI myelin metrics: magnetization transfer ratio (MTR)⁴, MT_sat⁵ (similar to MTR, but accounting for T1 effects), and the fractional pool size (F) obtained from quantitative MT⁶ (10-point sampling of the z-spectrum). To relate each metric to the absolute MVF, we used a combined MT/histology dataset in cynomolgous macaques as the ground truth⁷, with the scaling constants for F, MTR and MT_sat chosen to match the MVF from histology, assuming a zero intercept. First we computed correlations in brain parenchyma between the three myelin metrics. Then we calculated FVF using the NODDI model of diffusion⁸ ($$$FVF = MVF + AVF$$$), where AVF is the axon volume fraction, $$$AVF=(1-MVF)(1-v_{iso})v_{ic}$$$. Next, we computed the aggregate g-ratio with each of the three metrics, and looked at g-ratio differences between healthy white matter, normal-appearing white matter (NAWM) and MS lesions. Finally, we performed a theoretical computation varying the slopes (c) and intercepts (b) in the MVF model (MVF = c * MRI_metric + b) to see how improper MVF calibration would affect the aggregate g-ratio measurement. Figure 2 shows the MVF derived from MTR, MT_sat, and F in a healthy brain. The trends are similar, but the percent difference between healthy white and grey matter was 15.02% for MTR, 40.08% for MT_sat, and 45.86% for F. Figure 3 shows that F is better correlated with MT_sat than with MTR, partly due to the larger dynamic range of MT_sat, but also because the relationship between F and MTR in parenchyma is non-linear (MTR ~ T1 * F). Figure 4 shows aggregate g-ratio maps in the MS patient computed using MTR, MT_sat and F. When using F or MT_sat, the aggregate g-ratio was 0.76 in NAWM and 0.8 in lesions (p < .001). When using MTR, the g-ratio trend was reversed (0.76 in NAWM and 0.65 in lesions, p < .001). This reversed trend (when using MTR) indicates thicker myelin in lesions, which is highly unlikely. Figure 5 shows how improper MVF calibration can bias the g-ratio measures via the FVF metric.Most MT-based MVF mapping techniques have been linearly correlated with histology^9-12, particularly in MS brain, where the dynamic range is larger due to pathology. However, the literature suggests that the slope and intercept are acquisition dependent, and that neither linearity nor monotonicity is guaranteed^13,14. Figure 5 shows how important it is to get an accurate MVF estimate, even if the relationship is linear. Being off by a small amount will result in interpreting FVF changes as g-ratio changes, and vice versa. When using MTR as a surrogate for myelination, nonlinearity and the conflicting effects of magnetization transfer and T1 changes severely confound quantitative g-ratio computation. In healthy and normal-appearing white matter the g-ratio is relatively constant, so improper MVF calibration gives reasonable values. However, in lesions MTR drops much less than F (15% vs. 50%), whereas AVF drops by more. This makes it seem like little myelin was lost (despite large losses), so the result is an artifactually lowered g-ratio. On the other hand, MT_sat produces similar percent differences to qMT in white matter and MS lesions, making it a clinically feasible candidate for g-ratio mapping (one quarter of the qMT scan time and only 25% longer than conventional MTR).The result of an incorrect MVF calibration is not simply loss in sensitivity to g-ratio changes, but rather g-ratio trends that are statistically significant, incorrect, and highly dependent on the fiber volume fraction changes. MT_sat and F seem to be better suited for g-ratio mapping (compared to MTR), but great care must be taken in interpreting aggregate g-ratio values in light of the sensitivities demonstrated in this abstract.