Based on previous own work (1), the main objective of this report was to further improve visualization and delineation of human brainstem substructures in-vivo. The study was approved by the local ethics board. Informed consent was obtained from six healthy subjects (mean age: 26, range: 22-29, four female). All experiments were performed on a 7T MRI system (Achieva, Philips Healthcare, Cleveland, OH, USA) using a 32 channel receive coil (Nova Medical, Wilmington, DE). An inversion recovery 3D MPRAGE sequence was acquired: FOV: 230 x 230 mm², TR for Inversion: 5 s, TR: 7.9 ms, TE: 3.6 ms, flip angle: 5°, voxel size: (600μm)³, 90 slices, 1 signal average, SENSE factor 2.0, Bandwidth: 202 Hz/pixel, acquisition time: 09:05 minutes. The acquisition delay was set to 970 ms for gray matter nulling. This scan was repeated four times with the same settings which resulted in a total scan time of approximately 36:20 minutes. To account for displacement of anatomy, intra-subject rigid body realignment over the repetitions was performed. Subsequently the average of the four aligned datasets was calculated. Second, a 3D T₂*-weighted multi-gradient-echo (6 echoes) sequence was acquired: FOV: 230 x 230 mm², TR: 60 ms, first TE: 5.5 ms, delta TE: 6.0, flip angle: 18°, voxel size: (600μm)³, 90 slices, 1 signal average, SENSE factor 2, Bandwidth: 518 Hz/pixel, acquisition time: 16:26 minutes. The multi-echo MR signal intensity as a function of the echo time, $$$S(TE)$$$, was fitted pixel wise to the expression $$$lnS(TE)=lnS(TE=0)-R_2^**TE$$$ in a linear least-squares polynomial regression. T₂* was obtained by inversion: $$$T_2^*=\frac{1}{R_2^*}$$$. Finally the first four echo images of the T₂* weighted sequences were combined to a single image according to the concept of multiple-echo data image combination (MEDIC). The images were visually compared to histology and post-mortem MR images from the Duvernoy's Atlas (2) and Olzsewski and Baxter's Cytoarchitecture of the Human Brainstem (3).Displacements during the MPRAGE acquisitions were minute for in plane movement with maximal shifts of 0.2 mm (X-axis), 0.3 mm (Y-axis) but substantial in the Z-axis with maximal shifts of 1.8 mm. Rotation of the head was below one degree in the jaw angle with mean values of max. rotation of 0.48° ± 0.36°. The rotational changes in the pitch and roll angle were negligible (Fig. 1). Figure 2 shows the reformatted sections in three orientations of the isotropic MPRAGE dataset of one subject. Figure 4 illustrates all four contrasts of one subject at three levels of the brainstem (midbrain, pons, medulla oblongata) without annotation. Brainstem substructures were manually outlined by comparing to histology sections from the Duvernoy Atlas, as illustrated in figure 3 and 5. The identifiable structures were assigned as follows: ¹substantia nigra ²olivary nucleus, ³superior colliculus, ⁴inferior colliculus, ⁵medial lemniscus, ⁶mammillary body, ⁷red nucleus, ⁸decussation of the superior cerebellar peduncle, ⁹medial longitudinal fasciculus, ¹⁰mamillothalamic tract, ¹¹posterior commissure, ¹²periaequaductul gray matter, ¹³lateral lemnisucs, ¹⁴optic tract, ¹⁵superior cerebellar peduncle, ¹⁶ventral pontine decussation, ¹⁷inferior and lateral vestibular nuclei / spinal trigeminal nuclei and tract, ¹⁸pyramidal tract, ¹⁹medial longitudinal fasciculus / tectospinal tract, ²⁰inferior cerebellar peduncle, ²¹thalamic fasciculus, ²²lenticular fasciculus, ²³corticospinal tract, ²⁴spinal trigeminal nucleus and tract, ²⁵raphe nuclei, ²⁶pontine fibers.The isotropic MPRAGE sequence in the gray matter nulling regime provides an ideal contrast to identify small substructures within pons and medulla regions. T₂* and R²* maps, not affected by inhomogeneity of the transmit field (B₁⁺) and varying receive-coil sensitivity profiles provide a favorable contrast for structures within the midbrain area. Image combination based on MEDIC resulted in improved SNR and enhanced T₂* weighting. This facilitates the visualization of certain brainstem substructures hardly visible on the basis of MPRAGE/ T₂*/R₂* maps.Combining a segmented high resolution isotropic 3D MPRAGE in the gray matter nulling regime with T₂*/R₂* maps and MEDIC calculation resulted in improved visualization and differentiation of the complex human brainstem architecture in-vivo. Further effort is needed to adapt such an approach for standard clinical applications, particular including an optimized measurement time.