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Hi I loaded the seed and the targets in FSLView and they look correct and they are in FreeSurfer space. Is that correct? Or should I map them to dwi space and then perform tracking? No that’s correct, everything should be in freesurfer conformed space. None of the tracts reach none of the targets. fdt paths(in FreeSurfer space) has a small connectivity near thalamus but it ends near the thalamus. Bedpostx directions look fine. Do you suggest I should drop --usef argument? Yes definitely drop the —usef argument! Sorry I didn’t spot that. I suggest you drop all things that may eliminate or terminate streamlines for debugging and add them later on once you are happy that things are working. Saad Thanks Regards. On Wed, May 23, 2018, 6:51 AM Saad Jbabdi <[log in to unmask]<mailto:[log in to unmask]>> wrote: Hi This all looks sensible and thus it’s hard to know what went wrong without looking more closely at the output. Have you loaded all seed/target masks as well as fdt_paths onto fslview to check that everything is in the same space and that your streamlines indeed go through your target masks? Cheers Saad On 23 May 2018, at 02:56, neuroimage analyst <[log in to unmask]<mailto:[log in to unmask]>> wrote: Hi We are interested in generating connectivity between the thalamus and the rest of cortical hemisphere in each brain. In order to do so, I performed the following steps: a) Extract thalamus mri_extract_label $SUBJECTS_DIR/T1/T1_subject_ID_Baseline/mri/aseg.mgz 10 $SUBJECTS_DIR/subject_ID /labels/lh_thalamus.nii.gz b) Extract the left hemispheric labels mri_label2vol --label $label --temp $SUBJECTS_DIR/T1/T1_subject_ID_Baseline/anat/fs.nii.gz --o $vol --identity --fillthresh 0.5 > /dev/null echo $vol >> target.txt where fs.nii.gz is the freesurfer space T1 generated as: mri_convert $SUBJECTS_DIR/subject_ID/mri/orig.mgz anat/fs.nii.gz c) Create waypoints.txt mri_binarize --i $SUBJECTS_DIR/subject_ID/mri/aparc+aseg.mgz --match 2 --o anat/wm_lh.nii.gz mri_binarize --i $SUBJECTS_DIR/subject_ID/mri/aparc+aseg.mgz --match 41 --o anat/wm_rh.nii.gz This is the content of waypoints.txt anat/wm_lh.nii.gz anat/wm_rh.nii.gz d) Create transformation matrix from FreeSurfer space to DWI space fs2str=subject_ID.bedpostX/xfms/fs2str.mat str2fs=subject_ID.bedpostX/xfms/str2fs.mat fa2fs=subject_ID.bedpostX/xfms/fa2fs.mat fs2fa=subject_ID.bedpostX/xfms/fs2fa.mat fa2str=subject_ID.bedpostX/xfms/fa2str.mat str2fa=subject_ID.bedpostX/xfms/str2fa.mat # Register FreeSurfer Space T1 to acquired T1 tkregister2 --mov $SUBJECTS_DIR/T1/T1_subject_ID_Baseline/anat/fs.gz --targ $SUBJECTS_DIR/T1/T1_subject_ID_Baseline/anat/str.gz --regheader --reg /tmp/junk --fslregout $SUBJECTS_DIR/$fs2str --noedit # Invert convert_xfm -omat $SUBJECTS_DIR/$str2fs -inverse $SUBJECTS_DIR/$fs2str # Transform FA to structural flirt -in $SUBJECTS_DIR/subject_ID/dtifit_FA.gz -ref $SUBJECTS_DIR/T1/T1_subject_ID_Baseline/anat/str.gz -omat $SUBJECTS_DIR/$fa2str -dof 6 # Invert convert_xfm -omat $SUBJECTS_DIR/$str2fa -inverse $SUBJECTS_DIR/$fa2str # Concatenate and Inverse convert_xfm -omat $SUBJECTS_DIR/$fa2fs -concat $SUBJECTS_DIR/$str2fs $SUBJECTS_DIR/$fa2str convert_xfm -omat $SUBJECTS_DIR/$fs2fa -inverse $SUBJECTS_DIR/$fa2fs e) Perform tracking probtrackx2 -x $SUBJECTS_DIR/subject_id/labels/lh_thalamus.nii.gz -s $SUBJECTS_DIR/ subject_id.bedpostX/merged -m $SUBJECTS_DIR/ subject_id.bedpostX/nodif_brain_mask -l --usef --s2tastext --os2t --onewaycondition -c 0.2 -S 2000 --steplength=0.5 -P 5000 --fibthresh=0.01 --distthresh=0.0 --sampvox=0.0 --xfm=$SUBJECTS_DIR/ subject_id.bedpostX/xfms/fs2fa.mat --avoid=$SUBJECTS_DIR/T1/T1_subject_id _Baseline/anat/ventricles.nii.gz --seedref=$SUBJECTS_DIR/T1/T1_subject_id_Baseline/anat/fs.nii.gz --forcedir --opd -V 1 --omatrix1 --dir=$SUBJECTS_DIR/subject_id/lh_thalamus.probtrackx2 --waypoints=$SUBJECTS_DIR/T1/T1_subject_id_Baseline.nii/waypoints.txt --waycond='OR' --targetmasks=$SUBJECTS_DIR/subject_id/target.txt --meshspace=freesurfer However, the content of matrix_seeds_to_all_targets generated from the above line is all 0 and the waytotal has a value of 218681 with # of seed voxels being 7715. Can anyone comment what is the mistake and how can I generate left thalamus to left hemisphere connectivity with the seed and the targets in FreeSurfer space? Thanks