Identifying Parkinson's disease (PD) in its early stage is crucial to prevent its progression.¹ Several studies have reported on the significant and selective iron accumulation with diminishing neuromelanin cell (NMC) populations within substantia nigra (SN) of the midbrain from neurodegeneration.² Consequently, the hypointensity of T2* weighted image (WI) of SN is normally considered as the signature of iron deposition with aging brain.³ In this study, we systemically compared relaxation-derived T2* values and phase-derived quantitative susceptibility mapping (QSM) in postmortem ex vivo SN of normal subjects. Registered Kluver-Barrera and Perl's Prussian blue stained slides were used as a gold standard in localizing the iron and NMC distribution within SN and directly correlated with T2* and QSM-derived susceptibility values with varying MRI spatial resolution. Postmortem 40-year-old male and 70-year-old female brain tissues were fixed in 4% formalin at least 2 months for the stabilization. The brain was sectioned into 1.5cm-thick slices containing one-half of SN. MRI experiments were performed using Bruker 7T system. T1 weighted images were acquired using the fast spin echo sequence with TR = 700 ms, TE = 8 ms, and the magnetization transfer pulse (flip angle = 117, 1500 Hz off-resonance). The 2D multi-gradient echo sequence was used to obtain T2* maps and magnetic susceptibility maps with repetition time (TR) = 2000ms, the echo time (TE) = 3.1 ~ 40 ms (10 echoes, increment = 4.1 ms), and flip angle = 30 ̊. The following parameters were used in common: FOV = 35 x 35(mm), matrix size 256 x 256 thickness = 0.5 mm, and number of slices =20. The magnetic susceptibility map was reconstructed by quantitative susceptibility mapping (QSM) of phase information and R2* (1/ T2*) map was generated by linear fitting of relaxation data. After MRI scanning, 10 consecutive 50 μm-thick slices were sectioned, which corresponded to each MRI slice. The 4 serial slices were stained with Perl's Prussian blue staining to stain ferric iron, Kluver-Barrera staining which is sensitive to myelin with Nissl counterstain, TH immunohistochemistry to identify dopamine cells and fibers, and Calbindin D28K immunohistochemistry to subdivide the SN. Olympus virtual slide microscope (Olympus, Japan) was used to scan all stained slides with a pixel size of 0.6836 um² (100×). The density map (%) introduced the number of pixels occupied by neuron, NMC, and iron per 10 × 10 blocks extracted from each stained image. The MRI slices and corresponding stained images were co-registered for the same spatial resolution to compare each other on the pixel by pixel basis. The correlation coefficient was evaluated among 4 variables; R2*, magnetic susceptibility, iron density, and NMC density. Histograms for T2* and susceptibility values were generated for the NMC, iron and tissue regions, which were segmented from corresponding histology slices. Kluver-Barrera and Perl's Prussian blue stained slices were shown in Fig 1A and Fig 1B for spatial localization of SN region and direct visualization of iron and NMC. The hypointensity T2* -WI was observed within SN (Fig 1C) for elevated iron and NMC region, also correlated with lowered T2* values (Fig 1D) and increased susceptibility values from QSM (Fig 1E). The NMC, iron, and tissue areas segmented from KB and Perl's Prussian blue staining were used to differentiate T2* and susceptibility values for each region. The T2* values of iron occupied regions (mean = 9.90ms) and NMC occupied regions (mean = 9.93ms) were shorter than that of tissue region (mean = 10.97ms) in as shown in Fig 2. Similarly, the QSM derived susceptibility values of iron occupied regions (mean = 0.0711) and NMC occupied regions (mean = 0.0718) were larger than that of tissue region (mean = 0.0554) in as shown in Fig 3. We observed reduced T2* values and increased susceptibility values in the highly concentrated region of iron and NMC within the SN for normal subjects at 7T MRI. The origin of these perturbations, especially from NMC should be cautioned and requires further investigation for normal and PD patients in high field MRI diagnosis.