Abnormal iron level in the brain of Parkinson’s disease (PD) was postulated to be a cause of degeneration of dopamine-generating neurons in substantia nigra pars compacta^1-3. Studying of in vivo brain iron distribution is essential for the study of prognosis of the disease. In this study, we aimed to investigate the distribution of iron, in terms of measuring magnetic susceptibility with quantitative susceptibility mapping (QSM) technique, in the brain of PD patients particularly in the region of substantia nigra. Both voxel-wise and ROI-based studies were performed to obtain a better understanding to iron deposition in the PD brain.55 PD patients (37 males, mean age ± S.D. = 66 ± 9 years, mean illness duration ± S.D. = 9 ± 6 years) and 26 healthy control (15 males, mean age ± S.D. = 62 ± 7 years) were recruited. All QSM raw images were acquired using a 3.0T Philips scanner with the following parameters: 3D-T1FFE sequence, TR/TE = 28/23 ms, flip angle = 15°, NEX = 1, FOV = 230 x 230 x 180 mm³, reconstructed resolution = 0.45 x 0.45 x 1 mm³. QSM images were generated with the following approaches: Lapalcian-based phase unwrap, then background field removal with ReSHARP, followed by total variation regularization-based L1-norm QSM algorithm^4,5. The reconstructed QSM images were spatial normalized to MNI152 standard space with the FSL-FNIRT algorithms (fig. 1), and smoothed with a 3D-Gaussian filter before passing to SPM12 for voxel-wise analysis. For structural voxel-wise study, subcortical structures were either segmented by the FSL-FIRST (caudate, putamen, pallidum, thalamus, hippocampus and amygdala) or manually defined on the standard space (substantia nigra, red nucleus and dentate nucleus, fig. 1). The ROIs were applied as explicit masking during statistical analysis. To further analyze the susceptibility distribution in bilateral substantia nigra, slice-by-slice ROI comparison was performed. The measured susceptibility values were normalized to the averaged values of the bilateral frontal white matter. Two-sample t-test, adjusted for age and gender of the subjects, was employed in statistical analysis.Age and gender were included as covariates in the study for adjustment in the statistical tests. Whole-brain voxel-wise study with two-sample t-test showed that possible higher magnetic susceptibility in the bilateral substantia nigra in the PD brain (uncorrected p-value < 0.001, fig.2). In order to test for significance in individual subcortical structures, explicit masking was applied based on the ROI generated. Structural voxel-wise analysis of substantia nigra with two-sample t-test showed that some of the voxels in the structure, which is anatomically corresponded to the substanita nigra pars compacta, indicated a significantly higher magnetic susceptibility in PD patients (FEW-correct p-value < 0.05, fig.3). Statistical tests were also performed in other structures (bilateral thalamus, caudate, putamen, pallidum, hippocampus, amygdala, red nucleus and dentate nucleus) but yet no significance voxels were identified. Further analysis of substantia nigra by slice-by-slice ROI measurement in the standard space was carried out. The average susceptibility value was higher in the PD group, and some of the slices were tested to be statistically significant (p < 0.05, fig. 4). These slices also corresponded to the location of significant voxels in the structural voxel-wise study.Accumulation of mineral iron in substantia nigra of PD patients was extensively studied and reported with both post-mortem and in vivo approaches^1-3. The result of this study confirmed that substantia nigra, in particular the pars compacta, is the region with significantly higher iron loading in the PD brain. Both structural voxel-wise analysis and split ROI studies pointed to the same findings and the result agreed with each other. Non-invasive in vivo imaging of mineral iron with the QSM technique could be a potential tool for the prognostic study of the PD. This study, apart from reporting the abnormal iron level in substantia nigra pars compacta, also explored the feasibility of performing voxel-wise analysis. Traditional method to analyze substantia nigra iron concentration includes manual drawing of ROI on subject’s magnitude images or even the QSM maps by researchers. However, such work could be tedious in a large scale study, and introduction of bias and human error would be inevitable. Voxel-wise analysis can be completely automated, which reduces the subjective human error being introduced in the analysis. The method employed in this study, however, assumed accurate deformation of the subcortical structures with FSL. Check of registration accuracy is therefore essential prior to statistical analysis.Both voxel-wise and ROI-based analyzes confirmed higher iron concentration in substantia nigra pars compacta of PD patients. QSM could be a potential tool to study in vivo iron deposition in PD.