Evidence of White Matter R 2 * Homogenization After Formalin Fixation

ABSTRACT Purpose Disentangling intrinsic susceptibility‐driven R 2 * characteristics in white matter (WM) from fixation‐related effects using in vivo, postmortem (PM) in situ, and formalin‐fixed PM ex situ MRI. Methods DTI and multi‐echo gradient‐echo MRI were acquired in vivo, PM in situ and PM ex situ. PM cases included neurologically healthy individuals and patients with amyotrophic lateral sclerosis (ALS). Voxel‐wise R 2 * values were obtained using a mono‐exponential fit and evaluated both as whole WM averages and according to WM fiber‐to‐field orientation. For the PM ex situ orientation‐dependency analysis, PM ex situ R 2 * data were registered to the corresponding in situ DTI acquisition. Averaged as well as orientation‐dependent R 2 * profiles were pooled across WM and compared across conditions. Results Whole WM averaged and orientation‐dependent R 2 * values differed across in vivo, PM in situ, and PM ex situ conditions. No differences were observed between PM in situ healthy and PM in situ ALS cases. Compared with in vivo measurements, both PM conditions showed elevated whole WM averaged R 2 * values and reduced orientation dependency, with the strongest R 2 * anisotropy in vivo and nearly absent orientation dependency in the PM ex situ condition. Conclusion While orientation‐dependent R 2 * is reduced but preserved in unfixed PM in situ tissue, it is largely eliminated after prolonged formalin fixation without rehydration, likely due to microstructural reorganization and protein cross‐linking. Collectively, these findings indicate that orientation‐dependent R 2 * measured in fixed tissue has limited translational validity, while underscoring its sensitivity to intact microstructural organization in vivo and in unfixed PM tissue.