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Background: Tractography has been widely adopted to improve brain gliomas' surgical planning and guide their resection. This study aimed to evaluate state-of-the-art of arcuate fasciculus (AF) tractography for surgical planning and explore the role of along-tract analyses in vivo for characterizing tumor histopathology. Methods: High angular resolution diffusion imaging (HARDI) images were acquired for nine patients with tumors located in or near language areas (age: 41 ± 14 years, mean ± standard deviation; five males) and 32 healthy volunteers (age: 39 ± 16 years; 16 males). Phonemic fluency task fMRI was acquired preoperatively for patients. AF tractography was performed using constrained spherical deconvolution diffusivity modeling and probabilistic fiber tracking. Along-tract analyses were performed, dividing the AF into 15 segments along the length of the tract defined using the Laplacian operator. For each AF segment, diffusion tensor imaging (DTI) measures were compared with those obtained in healthy controls (HCs). The hemispheric laterality index (LI) was calculated from language task fMRI activations in the frontal, parietal, and temporal lobe parcellations. Tumors were grouped into low/high grade (LG/HG). Results: Four tumors were LG gliomas (one dysembryoplastic neuroepithelial tumor and three glioma grade II) and five HG gliomas (two grade III and three grade IV). For LG tumors, gross total removal was achieved in all but one case, for HG in two patients. Tractography identified the AF trajectory in all cases. Four along-tract DTI measures potentially discriminated LG and HG tumor patients (false discovery rate < 0.1): the number of abnormal MD and RD segments, median AD, and MD measures. Both a higher number of abnormal AF segments and a higher AD and MD measures were associated with HG tumor patients. Moreover, correlations (unadjusted p < 0.05) were found between the parietal lobe LI and the DTI measures, which discriminated between LG and HG tumor patients. In particular, a more rightward parietal lobe activation (LI < 0) correlated with a higher number of abnormal MD segments ( R = -0.732) and RD segments ( R = -0.724). Conclusions: AF tractography allows to detect the course of the tract, favoring the safer-as-possible tumor resection. Our preliminary study shows that along-tract DTI metrics can provide useful information for differentiating LG and HG tumors during pre-surgical tumor characterization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zoli, Talozzi, Martinoni, Manners, Badaloni, Testa, Asioli, Mitolo, Bartiromo, Rochat, Fabbri, Sturiale, Conti, Lodi, Mazzatenta and Tonon.)