344 Neuroinform (2012) 10:341–350 Fig. 1 Stereological and FreeSurfer methods used to estimate thalamic volume shown at approximately the same axial sections. Both methods are shown for the same hemisphere (the FreeSurfer axial sections are flipped to show maximal correspondence between techniques) of the same randomly selected subject. The sagittal MR section illustrates the an observer-independent approach, which could be con- trasted with the manual stereological measurements of the thalamus. Thalamic segmentations are based on the assign- ment of neuroanatomical labels to each voxel in an MR image based on the probabilistic information automatically estimated from a manually labelled training set. The meth- ods of the automated volumetric approach have been de- scribed in detail previously (Fischl, et al. 2002), and the accuracy of automated labelling and volumetry of subcorti- cal structures have been independently validated with re- spect to ‘gold standard’ manual volumetric techniques, predominantly for the hippocampus (Cherbuin, et al. 2009; Dewey, et al. 2010; Morey, et al. 2009; Pardoe, et al. 2009; Shen, et al. 2010; Tae, et al. 2008), and also of the amgydala (Dewey, et al. 2010; Morey, et al. 2009) and striatum (Dewey, et al. 2010). To our knowledge, there has been no independent comparison of the automated thalamic volumetry offered by FreeSurfer and a manual volumetric method (al- though thalamic tracings were compared with the perfor- mance of FreeSurfer in the original methods paper by Fischl et al. (2002)). Figure 1 shows the comparison of automated labelling of the thalamus (and extra-thalamic structures) in an individual control subject using FreeSurfer relative to stereo- logical volume estimation of the thalamus in the same subject. FreeSurfer analyses were performed on a Mac Pro (Version OS X 10.6.6, 32 GB, 2×2.93 GHz 6-Core Intel Xeon (HT)), which permitted the FreeSurfer ‘recon-all’ function (for corti- cal reconstruction and brain segmentation; http://surfer. nmr.mgh.harvard.edu/fswiki/recon-all) to complete 23 partic- ipants in less than 20 h. After the ‘recon-all’ function, the neuroanatomical labels were inspected for accuracy in all patients and controls. Despite that FreeSurfer permits manual editing to improve subcortical segmentation, no obvious errors in the automatic labelling were observed for any sub- ject, and so all data obtained from FreeSurfer analyses were 100% automated and not influenced by manual intervention. approximate levels of the axial sections shown. Abbreviations: di, ventral diencephalon (peach); pa, pallidum (violet); put, putamen (li- lac); th, thalamus (dark green); thp, pulvinar of the thalamus (dark green) Statistical Analyses Two-way mixed intra-class correlation coefficients for ab- solute agreement (Shrout and Fleiss 1979) were used to determine inter-rater agreement between two human raters using manual stereology and FreeSurfer for volume estima- tion of the thalamus in ten randomly selected controls using the statistics software SPSS (Version 18, www.spss.com). Intra-class correlations were subsequently performed be- tween stereological volumes obtained by one human rater and FreeSurfer volumes for the entire sample of patients and Fig. 2 Inter-rater consistency in stereological volume estimation of the left (blue) and right (red) thalamus, and relation to volume estimates obtained from FreeSurfer. Stereological volume estimates are repro- ducible, and FreeSurfer is entirely consistent with the volumes obtained with both rater one and rater two. Error bars indicate the 95% confidence intervals