Myelin Measurement: Comparison Between Simultaneous Tissue Relaxometry, Magnetization Transfer Saturation Index, and T1w/T2w Ratio Methods

Magnetization transfer (MT) imaging has been widely used for estimating myelin content in the brain. Recently, two other approaches, namely simultaneous tissue relaxometry of R1 and R2 relaxation rates and proton density (SyMRI) and the ratio of T1-weighted to T2-weighted images (T1w/T2w ratio), were also proposed as methods for measuring myelin. SyMRI and MT imaging have been reported to correlate well with actual myelin by histology. However, for T1w/T2w ratio, such evidence is limited. In 20 healthy adults, we examined the correlation between these three methods, using MT saturation index (MTsat) for MT imaging. After calibration, white matter (WM) to gray matter (GM) contrast was the highest for SyMRI among these three metrics. Even though SyMRI and MTsat showed strong correlation in the WM (r = 0.72), only weak correlation was found between T1w/T2w and SyMRI (r = 0.45) or MTsat (r = 0.38) (correlation coefficients significantly different from each other, with p values < 0.001). In subcortical and cortical GM, these measurements showed moderate to strong correlations to each other (r = 0.54 to 0.78). In conclusion, the high correlation between SyMRI and MTsat indicates that both methods are similarly suited to measure myelin in the WM, whereas T1w/T2w ratio may be less optimal.

images separately 10 . At the same time, automatic brain segmentation 11 and myelin measurement 12 are also possible using the acquired quantitative values. These can be done with a dedicated software called 'SyMRI' with post-processing time less than 1 minute 9 . Thus, myelin measurements can now be performed within the limits of clinically allowed scanning time. The myelin model infers myelin volume fraction (MVF) in a voxel based on the effect of myelin on intra-and extracellular water relaxation rates due to magnetization exchange 12 . The observed R 1 and R 2 rates of intra-and extracellular water increase in the vicinity of fast relaxing myelin water. On the other hand, the observable PD decreases because myelin water decays much faster than non-myelin water. The SyMRI myelin measurement has been validated on 12 human cadavers using Luxol Fast Blue staining of histological specimens 13 . A repeatability study has reported a very low error (coefficient of variation, 0.59% for 0.8 mm in-plane resolution) for whole-brain myelin volume calculated using SyMRI 14 . Myelin volume measured by SyMRI has been shown to depend on age in pediatric populations, especially in children under 4 years old, thus indicating a correlation of this method with the normal myelination process 15,16 . This method has also been used in studies investigating patients with multiple sclerosis (MS) 17,18 , Sturge-Weber syndrome 19 , and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) 20 , with promising results. However, correlation of SyMRI myelin measurement with other MRI techniques sensitive to myelin has not been investigated so far.
There are several other techniques for myelin measurement, including myelin water imaging 21,22 , macromolecular tissue volume derived from normalized PD mapping 23 , and magnetization transfer (MT) imaging 2 . MT is a phenomenon where the proton spins bound to macromolecules, once excited by a radiofrequency pulse, transfer a part of their energy to the neighboring mobile proton spins 24 . MT imaging estimates the macromolecular proton pool size with ultra-short T 2 relaxation by transfer of magnetization to the observable mobile water pool 25 . MT ratio (MTR) has been widely used based on this theory and shown to correlate well with histological myelin content 26,27 , but also with other properties such as R 1 24 . R 1 also correlates strongly with myelin 28 , meaning that MTR and R 1 work against each other and R 1 mitigates the power of MTR as a measure of myelin. Further, R 1 is also sensitive to iron, calcium content, and axon size 29 and count 30 , thus making the relationship between MTR and actual myelin content nonlinear. MT saturation (MT sat ) imaging was developed to improve MTR, by decoupling MTR from R 1 31 . MT sat shows higher contrast in the brain than MTR does 31 , and has been shown to correlate more with disability metrics than MTR in patients with MS 32 . MT sat has also been shown to correlate well with quantitative MT measures 25 , which reduces dependency of MT imaging on sequence parameters. However, quantitative MT imaging is time-consuming and the post-processing is still challenging. T 1 w/T 2 w ratio is another approach for assessing myelin content in the cortical gray matter, originally developed to map myeloarchitecturally distinct cortical regions for parcellation of cerebral cortex, thus providing a connectivity measurement 33,34 . Pixel intensity on T 1 w and T 2 w images is assumed to be directly and inversely proportional to myelin contrast, respectively. Thus, ratio of these images is thought to accentuate the intrinsic contrast of myelin. Because intensity scaling of T 1 w and T 2 w images differ across scanners and acquisition protocols, Ganzetti et al. 35 have suggested that calibration of their intensities prior to making their ratio can increase the reproducibility of T 1 w/T 2 w ratio. Although T 1 w/T 2 w ratio is not a direct index of myelin, it is still considered a proxy of myelin content 36 . While intracortical myelin content across different ages has been evaluated using this method 36,37 , myelination of white matter (WM) in neonatal brains has also been investigated using this method 38,39 . Further, the test-retest reliability of T 1 w/T 2 w ratio has been reported to be high 40 . Recent histological studies investigated T 1 w/T 2 w ratio in patients with MS, showing that T 1 w/T 2 w ratio was significantly different between myelinated and demyelinated cortex in MS patients 41 , and also significantly different in the cortex between early-stage MS and healthy controls 42 . Because T 1 w and T 2 w images are routinely acquired as part of brain MRI protocols, this technique does not increase scanning time. However, the specificity of T 1w /T 2w to actual myelin content has been doubted by recent studies 40,43 .
As mentioned above, there are several different methods to estimate myelin volume in the brain. However, investigation of correlation among different methods is scarce. Specifically, no study has examined the correlation of SyMRI as a myelin imaging tool with other methods. Therefore, the aim of this study was to compare SyMRI with two other putative myelin measurement techniques by investigating the correlation of SyMRI with MT sat and T 1 w/T 2 w ratio in WM and gray matter (GM).

Results
Scatterplots and Mean Values of MVF MTsat , MVF SyMRI , and MVF T1w/T2w . The calibration factors for MVF MTsat and MVF T1w/T2w were 8.40 and 14.5, respectively, so that their means in the WM equaled that of MVF SyMRI . The scatterplots of these three MVF metrics are shown in Fig. 1. Table 1 shows the mean and standard deviation (SD) of each MVF metric after calibration, and MT sat and T 1 w/T 2 w ratio before calibration in each tissue region, with the percentage of MVF in subcortical or cortical GM to that in WM. Because both MVF MTsat and MVF T1w/T2w were calibrated to MVF SyMRI , so that their mean values in the WM were equal, the mean values of WM for all these metrics were the same. The contrasts among WM and subcortical GM, and WM and cortical GM were significantly higher for MVF SyMRI and lower for MVF T1w/T2w than other MVF metrics (p < 0.001).
Correlation Coefficients among MVF MTsat , MVF SyMRI , and MVF T1w/T2w . Table 2 shows the Spearman's ρ correlation coefficients with their 95% confidence intervals (CIs) among MVF metrics. Correlations were significant for all regions-alone or combined-among these metrics (p < 0.001). In the WM and subcortical GM, the correlation coefficient was the highest between MVF MTsat and MVF SyMRI (p < 0.001 in the WM and p = 0.005 in the subcortical GM). In the WM, MVF T1w/T2w showed only weak to moderate correlation with MVF MTsat or MVF SyMRI . In the cortical GM, the correlation coefficient was the highest between MVF SyMRI and MVF T1w/T2w (p < 0.001), with MVF MTsat vs. MVF T1w/T2w showing the lowest value (p = 0.011). In all regions combined, all these metrics showed strong correlations. Correlation coefficients of MVF MTsat vs. MVF SyMRI and MVF SyMRI vs. MVF T1w/T2w were comparable (p = 0.62) and higher than that of MVF MTsat vs. MVF T1w/T2w (p < 0.001) Table 3 shows the Spearman's ρ correlation coefficients among MVF metrics in individual areas representative of 10 WM, 2 subcortical GM, and 4 cortical GM, and their mean values. Out of 10 WM ROIs, 8 showed significant correlations between MVF MTsat and MVF SyMRI . The 2 WM ROIs that did not show significant correlation were genu and splenium of corpus callosum, which showed the highest MVF SyMRI . Meanwhile, only 3 and 4 ROIs showed significant correlation between MVF MTsat and MVF T1w/T2w , and MVF SyMRI and MVF T1w/T2w , respectively. Both of the 2 subcortical GM ROIs showed significant correlations in all comparisons, with comparison between MVF MTsat   Table 4 shows the values of the intercept and slope with their standard error in each region-alone or combined-for MVF SyMRI and MVF T1w/T2w as a function of MVF MTsat . In WM, cortical GM, and all regions combined, significant difference was detected between the slopes of MVF SyMRI and MVF T1w/T2w , with that of MVF SyMRI nearer to 1. In subcortical GM, slopes of MVF SyMRI and MVF T1w/T2w did not show statistical significance, and y-intercepts differed significantly with that of MVF T1w/T2w nearer to 0.

Discussion
In this study, we investigated the concurrent validity of SyMRI myelin measurement method by comparing SyMRI with MT sat and T 1 w/T 2 w ratio in WM and GM. As part of the study, we tried to estimate the absolute myelin partial volume in a voxel by these three methods. SyMRI directly estimates MVF of a voxel by bloch simulation. On the other hand, MT sat and T 1 w/T 2 w ratio require calibration to be used as quantitative measures of myelin content. Thus, we calibrated MT sat and T 1 w/T 2 w ratio for their means in the whole WM to be equal to that of MVF SyMRI , partly because calibration method does not affect correlation coefficient and contrast between WM and cortical or subcortical GM. In this study, the mean MVF SyMRI in the WM was 30.70%. This corresponds to the previously reported values (around 25-30%) of MVF in WM, investigated by histology 2,44 . This value also corresponds to the results of MVF investigated using SyMRI for WM of cadavers (30.98%) 13 and normal-appearing WM of MS patients (32.88% and 30.96%) 17,18 . For GM, reports on investigation into MVF by histology are rather scarce and most were performed with optical density using Luxol Fast Blue stain, which could be used only in comparison with the values of other brain microstructures 45 . Previous studies that investigated volume fraction  of myelin in the brain showed optical densities of subcortical and cortical GM to be around 49-67% and 9.8-36% that of WM, respectively 13,46 . In our study, MVF SyMRI corresponded to the results of these histological studies in cortical GM better than MVF MTsat and MVF T1w/T2w . For subcortical GM, MVF MTsat and MVF SyMRI were comparable and these showed better correspondence to previous histological study than MVF T1w/T2w . In terms of WM to GM contrast, we conclude that MVF SyMRI was the best fit to the results of previous histological studies among the metrics investigated in our study. In our study, we investigated the correlation among three different metrics for myelin content. The aim was to show the concurrent validity of MVF SyMRI by MVF MTsat and MVF T1W/T2W . For WM, MVF SyMRI showed strong and higher correlation with MVF MTsat than MVF T1w/T2w . In regression analysis, the slope was closer to 1 for MVF SyMRI than MVF T1w/T2w as a function of MVF MTsat in WM. These results are in line with the study by Arshad et al. 40 . They investigated the correlation between T 1 w/T 2 w ratio and myelin water fraction in WM, and found that T 1 w/T 2 w ratio poorly correlated with myelin water fraction and correlated more with geometric mean of multi-echo T 2 relaxation, which had been shown to correlate with axon diameter based on histology, rather than myelin content 47 . Another study also showed poor correlation between T 1 w/T 2 w and myelin water fraction 43 . Therefore, T 1 w/ T 2 w ratio may not be a suitable candidate as a measure of myelin in WM. In cortical GM, these three MVF metrics showed moderate to strong correlations to each other, with MVF SyMRI and MVF T1w/T2w showing a higher correlation. However, we cannot determine which is the best measure for estimating myelin content in GM among these three metrics at this moment. Myeloarchitecture is different among cortical areas, and high-resolution T 1 w/ T 2 w ratio has been widely used for cortical parcellation, especially in the Human Connectome Project, showing good results 48 . In a future study, comparison of these metrics for the ability of cortical parcellation should be investigated. However, recent histological study showed that T 1 w/T 2 w ratio in the cerebral cortex correlated well with dendrites, but not with myelin, even though the sample size was small (9 MS patients) 42 . There is a possibility that T 1 w/T 2 w ratio does not reflect actual myelin content in the brain. All regions in aggregate showed strong correlation coefficients in all comparisons (i.e. MVF MTsat vs. MVF SyMRI , MVF MTsat vs. MVF T1w/T2w , and MVF SyMRI vs. MVF T1w/T2w ). This may be because subgroups with different microstructures were included in the analysis.
When we analyzed individual structures representative of WM, subcortical GM, and cortical GM, the correlation coefficients showed similar tendency to those shown for each segment as a whole. Of note, only genu and splenium of corpus callosum out of the 10 WM ROIs did not show significant correlation between MVF MTsat and MVF SyMRI , with these showing the highest MVF SyMRI . This may be because SyMRI does not assume nonphysiological MVF higher than 40% 12 , and disagreement may have occurred between SyMRI and MT sat with high values.
Determination of the precise relationship between MRI measures of myelin and actual MVF is especially important for calculating the g-ratio, which is the ratio of the inner and the outer diameter of a myelinated nerve fiber 49 . Calculation of the g-ratio by MRI can be performed with myelin imaging in combination with diffusion MRI, such as diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) 49,50 . Because diffusion MRI alone is not sufficient to estimate axon volume fraction 49 , precise measurement of myelin is necessary for correct g-ratio calculation. Furthermore, g-ratio could complement MVF measurements in understanding tissue microstructure, because MVF only cannot differentiate partial demyelination of neuronal fibers from loss of axons, with the remaining axons fully myelinated. Thus, g-ratio can provide a more complete picture of the microstructure, which is important for understanding plasticity of the normal brain 51 and may also be important for the care of patients with MS in choosing immunotherapy or remyelination therapy 25 . Because we could not perform histological measurements of actual myelin content in this study, we calibrated MT sat and T 1 w/T 2 w ratio to MVF SyMRI . Even though we assumed zero-intercept upon calibration of MVF MTsat and MVF T1w/T2w to MVF SyMRI , we detected a non-zero intercept when linear regression was performed. This means that at least two of these MVF metrics are not perfectly specific to myelin content in the brain. Although it may be expected that MT sat is also sensitive to macromolecules other than myelin, the specificity of our MVF metrics to actual myelin content should be investigated more precisely in future histological studies. We should also be aware that scaling factors depend on the acquisition protocol and post-processing, and should be carefully determined for each investigation 25 .
Rapid relaxation of myelin water cannot be directly measured by the SyMRI sequence, but the presence of MVF can be inferred by its effect of magnetization exchange with the slower cellular relaxation, as well as the decrease in observed PD. This is an indirect measurement and may have some limitations when compared with a more direct approach, such as myelin water fraction, which estimates T 2 distribution of water including myelin water by fitting multi-exponential T 2 decay 22 and has been shown to correlate well with histological myelin content in patients with MS 52 . However, for clinical use, the robustness and easy implementation may be more important. SyMRI myelin measurement has been shown to have good repeatability, which is important for longitudinal studies 14 . In addition to myelin measurements, any contrast-weighted image can also be generated by SyMRI 53 , thus obviating the need for further conventional scans. There are several limitations in this study. First, the resolutions of the images were different between MVF SyMRI or T 1 w/T 2 w ratio (2D acquisition) and MT sat (3D acquisition). Even though the difference in resolution could introduce deviation in the quantification, this would have been offset by a large number of ROIs used in this study. However, the analyses of 2D and 3D images by consistent methods was a challenge in our study. Rather than co-registering these images, we registered ROIs in template space to 2D or 3D space for each subject. Co-registration may cause some mis-registration, which will result in inappropriate comparison of voxels derived from different tissues. When we applied the ROIs to each MVF map, we used partial volume maps of GM, WM, or both, with thresholding, to minimize partial volume effects. Second, T 1 -weighted images for T 1 w/T 2 w ratio were acquired by a spin-echo sequence, even though mostly gradient-echo sequences have been used for calculating T 1 w/T 2 w ratio 33,35,36,40,48 . Because T 1 w/T 2 w ratio is a semi-quantitative value, different acquisitions may introduce different contrasts. However, T 1 w/T 2 w ratio has been shown to give very similar overall results when acquired on different scanners with different sequences and different field strengths 33,35 . Third, the myelin measurement methods investigated in this study may show variable behaviors in diseased brains from healthy brains, not only due to demyelination but also due to edema, inflammation, iron accumulation, or atrophy. This should be investigated in future studies. For example, MTR seems to correlate with not only myelin but also with change in water content caused by inflammation or edema in patients with MS 54 . Even though we assumed a linear relationship for calibration of MVF values, this assumption may not hold true in diseased brains.
In summary, we compared MT sat , MVF SyMRI , and T 1 w/T 2 w ratio as quantitative measures of myelin in the brain. We calibrated MT sat and T 1 w/T 2 w in WM to be equal to MVF SyMRI in WM (MVF MTSat and MVF T1w/T2w ). Correlation of these metrics in WM was strong and higher between MVF MTsat and MVF SyMRI than between MVF T1w/T2w and MVF MTsat or MVF SyMRI , indicating that MVF MTsat and MVF SyMRI are similarly suited to measure myelin in the WM, whereas MVF T1w/T2w may be less optimal. In GM, moderate to strong correlation was observed among these metrics. However, further studies performing cortical parcellation using these measures or investigating the correlation between each MVF metric and histology should be conducted before concluding which is the best measure for estimating myelin content in GM.

Materials and Methods
Study Participants. Twenty healthy volunteers (9 male and 11 female, mean age 55.3 years, age range 25-71 years) were included in this study. These subjects were screened by a questionnaire for neurological or psychological symptoms, or history of neurologic diseases. Acquired images were also screened for moderate-to-severe WM ischemic lesions (Fazekas grade 2 or more 55 ), asymptomatic cerebral infarction, or regional brain atrophy.
Ethical issue. All data from the patients were obtained in accordance with the 2013 revised Helsinki Declaration of 1964. We provided participants with detailed information, and written informed consent was obtained from all participants. The Ethical Committee of Juntendo University Hospital approved the study. MRI Acquisition Protocol for SyMRI. All subjects were scanned on a single 3T MRI scanner (MAGNETOM Prisma, Siemens Healthcare, Erlangen, Germany) using a 64-channel head coil. QRAPMASTER (an acronym derived from 'quantification of relaxation times and proton density by multi-echo acquisition of a saturation-recovery by using turbo spin-echo readout' for simultaneous tissue relaxometry) was performed for all subjects. QRAPMASTER is a two-dimensional (2D) axial multi-slice, multi-echo, and multi-saturation delay saturation-recovery turbo spin-echo acquisition method with which images are collected with different combinations of echo times (TEs) and saturation delay times. In our institution, combinations of 2 TEs and 4 delay times were used to make a matrix of 8 complex images that were then used to quantify longitudinal R 1 relaxation and transverse R 2 relaxation rates and PD by using SyMRI software 8.0 (SyntheticMR, Linköping, Sweden). The TEs were 22 and 99 ms, and the delay times were 170, 620, 1970, and 4220 ms. The repetition time (TR) was 4250 ms. The other parameters used for QRAPMASTER were as follows: field of view (FOV) 230 × 186 mm; matrix 320 × 260; echo-train length 10; bandwidth 150 Hz/pixel; parallel imaging acceleration factor 2; slice thickness/ gap 4.0 mm/1.0 mm; 30 sections; and acquisition time 5 min 8 sec.
Processing of SyMRI Data. Based on the R 1 , R 2 , and PD values acquired by QRAPMASTER, myelin volume fraction (MVF SyMRI ) was also calculated automatically on SyMRI software. This model for myelin measurement hypothesizes 4 compartments in the brain: myelin, cellular, free water, and excess parenchymal water partial volumes 12 . The model assumes that the relaxation behavior of each compartment contributes to the effective relaxation behavior of an acquisition voxel. The R 1 , R 2 , and PD values of free water and excess parenchymal water partial volumes were fixed to those of cerebrospinal fluid (CSF) (R 1 , 0.24 sec −1 ; R 2 , 0.87 sec −1 ; PD, 100%) 8 . The R 2 of myelin partial volume was fixed to the literature value of 77 sec −1 56 . Optimization of other model parameters were done by performing simulation by running Bloch equations for observable R 1 , R 2 , and PD properties in a spatially normalized and averaged brain from a group of healthy subjects 12 . In this model, the magnetization exchange rates between partial volume compartments are also considered. A lookup grid was made in R 1 -R 2 -PD space for all possible distributions (ranging from 0% to 100%) of the four partial volumes. The measured R 1 , R 2 , and PD values were projected onto the lookup grid, for estimating the MVF SyMRI in each voxel. Although other methods for myelin imaging require scaling factors to estimate MVF from measured macromolecular pool size or myelin water fraction, assuming linear proportionality 2 , we omitted this procedure because MVF SyMRI directly estimates the volume fraction of myelin in a voxel 12 . Processing of T1w/T2w ratio. Synthetic T 1 w and T 2 w images were produced from QRAPMASTER data.
Parameters used for T 1 w images were: TR 500 ms; and TE 10 ms. Parameters used for T 2 w images were: TR 4500 ms; and TE 100 ms. These T 1 w and T 2 w images were intrinsically aligned. Synthetic T 1 w and T 2 w images were skull-stripped using the intracranial mask generated by SyMRI software 57 . In conventional MRI, imperfection of B 1 field affects T 1 w and T 2 w images, generating intensity non-uniformity in these images. It has been proposed that this non-uniformity should be corrected before the ratio of these images is calculated, because a ratio does not adequately cancel the intensity non-uniformity 35 . The QRAPMASTER sequence acquires the B 1 field map and the acquired quantitative data are automatically corrected for local B 1 field when processed by SyMRI software 9 . Because T 1 w and T 2 w images are non-quantitative, the intensity scaling may vary among different individuals, sequences, or scanners. To minimize the effect of intensity scaling, we applied an external linear calibration to these contrast-weighted images as proposed by Ganzetti et al. 35 , which would provide a more consistent range of T 1 w and T 2 w intensities even across different datasets. Two masks of anatomical structures external to the brain-one with high T 1 w signal intensity and low T 2 w signal intensity (temporalis muscle) and the other with opposite properties (eye)-were used for calibration. These regions were defined in the MNI152 space using the ICBM152 template images (http://www.bic.mni.mcgill.ca/ServicesAtlases/ICBM152NLin2009) and then warped to each subject's space using the registration matrix described below in the ROI Analysis section. Distribution peaks (modes) of intensity values were recorded for these regions of interest (ROIs) in each subject. In ICBM152 template images, we recorded the modes as reference values for the eyes as following: 28.2 for T 1 w images and 99.9 for T 2 w images. For the temporalis muscle, the values were: 58.6 for T 1 w images and 21.1 for T 2 w images. The linear scaling of either T 1 w or T 2 w images was performed using the following equation 35 where I and I C represent the images before and after calibration. E S and M S are the mode intensity values of each subject's eye and muscle masks, respectively, and E R and M R show the reference values in template images of eye and muscle masks, respectively. After calibrating the T 1 w and T 2 w images, their ratio was calculated to produce the T 1 w/T 2 w ratio images.
Acquisition and Processing of MT sat . Three three-dimensional (3D) multi-echo fast low-angle shot (FLASH) sequences were performed with predominant T 1 -, PD-, and MT-weighting for all subjects. For T 1 w images, TR/excitation flip angle α = 10 ms/13° were used; for PD-and MT-weighted images, 24 ms/4° were used. For MT-weighted images, excitation was preceded by an off-resonance Gaussian-shaped RF pulse (frequency offset from water resonance 1.2 kHz, pulse duration 9.984 ms, and nominal flip angle 500°). For the other parameters, the following was used: slice thickness 1.8 mm; 104 slices; FOV 224 × 224 mm; matrix 128 × 128, parallel imaging using GRAPPA factor 2 in phase-encoding direction; 7/8 partial Fourier acquisition in the partition direction; bandwidth 260 Hz/pixel; and total acquisition time 6 min 25 sec. These three images were used to calculate the MT sat index 31 . First, the apparent longitudinal relaxation rate R 1app was calculated as follows: where S T1 and S PD denote signal intensities of T 1 w and PD-weighted images, respectively; TR T1 and TR PD denote TR of T 1 w and PD-weighted images, respectively; and α T1 and α PD denote excitation flip angles of T 1 w and PD-weighted images, respectively. Secondly, the apparent signal amplitude A app was calculated as follows: T1  PD T1 PD  T1 PD T1   T1  PD T1  PD  T1 PD Thirdly, the apparent MT saturation δ app was calculated as follows: where S MT , TR MT , and α MT denote signal intensity, TR, and excitation flip angle of MT-weighted image, respectively. The apparent MT saturation is inherently robust against differences in relaxation rates and inhomogeneities of RF transmit and receive field compared with conventional MTR imaging 31,58 . Furthermore, we also corrected for small residual higher-order dependencies of the MT saturation on the local RF transmit field to further improve spatial uniformity, as suggested by Weiskpof et al. 59 : 20° flip angles were acquired in short acquisition time (around 10 seconds each). The first image was acquired after excitation with a flip angle α and had a magnitude proportional to sin(α). The second image was acquired after excitation with a flip angle 2α and had a magnitude proportional to sin(2α). The ratio of the two acquisitions was formed giving: α α α = sin sin2 1 2 cos (6) from which the local flip angle α was calculated.
ROI Analysis. We used Johns Hopkins University (JHU) ICBM-DTI-81 WM labels atlas 61,62 and the automated anatomical labeling (AAL) atlas 63,64 to define WM and GM ROIs, respectively. The JHU ICBM-DTI-81 WM labels atlas comprised 48 WM ROIs; AAL comprised 116 ROIs including 12 subcortical GM ROIs. Even though MVF SyMRI and T 1 w/T 2 w ratio were in an identical space with the same resolution and slice thickness, MT sat had a different resolution and slice thickness. To ensure that ROIs were placed in the same anatomical position in these different spaces, we warped the above ROIs to each metric map. For generating the warp field to convert ROIs in the template space to each subject's space, we first used the FMRIB Software Library (FSL) linear and nonlinear image registration tool (FLIRT and FNIRT) 65,66 to register synthetic T 1 w and 3D T 1 w images to the MNI152 template. The generated warp fields were saved and inverted so they could be applied to all ROIs, including the eye and temporalis muscle masks. Next, to remove the partial volume effects from other tissues, we segmented synthetic T 1 w and 3D T 1 w images into WM, GM, and CSF using FMRIB's Automated Segmentation Tool (FAST) 67 . These segmented images of WM and GM were used as masks and applied to MVF SyMRI , T 1 w/T 2 w ratio, and MT sat . These tissue masks were thresholded at 0.95 to make sure that the masks contained WM or GM with a probability of 0.95 or higher. WM plus GM tissue masks were also made and thresholded at 0.95. For MVF SyMRI and T 1 w/T 2 w ratio, we used tissue masks based on the synthetic T 1 w images; for MT sat , we used tissue masks made from 3D T 1 -weighted images. For applying the ROIs from the JHU ICBM-DTI-81 WM labels atlas, we used MVF SyMRI , T 1 w/T 2 w ratio, and MT sat masked by WM tissue masks. For applying the ROIs from the AAL atlas to cortical GM, we used MVF SyMRI , T 1 w/T 2 w ratio, and MT sat masked by GM tissue masks. For applying the ROIs from the AAL atlas to subcortical GM (e.g., thalamus), we used MVF SyMRI , T 1 w/T 2 w ratio, and MT sat masked by GM plus WM tissue masks, because many parts of subcortical GM were segmented as WM by FAST. After warping, all ROIs were inspected for gross registration errors. Upon ROI analysis, the mean values were recorded for further analysis. Examples of ROI placement are shown in Fig. 2.

Calibration of MVF.
Even though SyMRI directly estimates MVF of a voxel, MT sat and T 1 w/T 2 w cannot be used as quantitative myelin markers as they are. For calibration of MT sat and T 1 w/T 2 w ratio to be used for quantifying myelin in the brain, we assumed a linear relationship between MVF SyMRI , MT sat , T 1 w/T 2 w ratio, and actual myelin content, as described previously for MT sat 68 . In the brain, not only myelin, but also other microstructures contribute to the values of MT sat and T 1 w/T 2 w ratio. However, if we assume a linear relationship between MT sat or T 1 w/T 2 w ratio and actual myelin content, MT sat or T 1 w/T 2 w ratio would also correlate linearly with non-myelin microstructures. Hence, the intercept of the regression line of actual myelin on MT sat or T 1 w/T 2 w would be near to zero. Since several studies have calibrated scaling factors of myelin sensitive metrics by healthy WM 25,49,68 , we also decided to calibrate MT sat and T 1 w/T 2 w ratio by values of WM. We determined the scaling factors of T 1 w/ T 2 w ratio and MT sat by making the means of these values in all the 48 WM ROIs equal to the mean MVF SyMRI . We denoted calibrated MT sat and T 1 w/T 2 w ratio as MVF MTsat and MVF T1w/T2w , respectively. Maps of MVF MTsat , MVF SyMRI , and MVF T1w/T2w are shown in Fig. 3. After calibration, we performed ROI analysis again for MVF T1w/ T2w and MVF MTsat as described in the previous section and mean values were recorded.

Statistical analysis.
For MVF values, normality was tested with the Shapiro-Wilk test. All of the datasets were not normally distributed; therefore, we used the Steel-Dwass test, which is a nonparametric test for multiple comparisons, to compare the contrast among WM and cortical GM, and WM and subcortical GM for the three MVF metrics, and used Spearman's rank order correlation coefficient to investigate the correlation among MVF metrics for WM, subcortical GM, and cortical GM. Spearman's ρ correlation coefficients were classified by using the following definitions: 0-0.30, very weak; 0.30-0.50, weak; 0.50-0.70, moderate; 0.70-0.90, strong; and 0.90-1.00, very strong 69 . Comparison of correlation coefficients among MVF MTsat vs. MVF SyMRI , MVF MTsat vs. MVF T1w/T2w , and MVF SyMRI vs. MVF T1w/T2w were performed in WM, subcortical GM, and cortical GM. This was performed with the Z test for the equality of the two correlations after Fisher r-to-Z transformation 70 . In addition to analyzing each segment as a whole, we also performed correlation analysis in individual structures representative of WM (genu of corpus callosum, splenium of corpus callosum, anterior limb of internal capsule, posterior limb of internal capsule, anterior corona radiata, superior corona radiata, posterior corona radiata, posterior thalamic radiation, external capsule, and superior longitudinal fasciculus), subcortical GM (pallidum and thalamus), and cortical GM (precentral, postcentral, Heschl, and lingual). Other than corpus callosum, we used bilateral regions aggregately in the analysis. Simple linear regression analysis was performed on the MVF SyMRI and MVF T1w/T2w as a function of MVF MTsat . The regression lines for MVF SyMRI and MVF T1w/T2w were compared by analysis of covariance to determine if they were significantly different from each other in WM, subcortical GM, cortical GM, and all regions combined. All statistical analyses were performed with the software package R, version 3.2.1 (http:// www.r-project.org/). A 2-sided p value < 0.05 was considered significant.  A and B) show transformed ROIs overlaid on 2D synthetic and 3D T 1 -weighted images in the same subject, respectively. Transformed ROIs for cortical GM and WM were masked by GM and WM partial volume maps thresholded at 0.95, respectively. For subcortical GM ROIs, GM plus WM partial volume maps thresholded at 0.95 were used for masking. For analysis, ROIs transformed to 2D synthetic T 1 -weighted images were applied to MVF SyMRI and T 1 w/T 2 w ratio, and ROIs transformed to 3D T 1 -weighted images were applied to MT sat .