346 FreeSurfer thalamic volumes were similarly smaller in patients relative to controls in the left (F(1,70)0 4.76, p0 0.03) and right (F(1,70)0 7.36, p0 0.008) hemispheres. Discussion The volume of the thalamus is a notoriously difficult metric to estimate reliably given the low contrast between thalamic gray matter and adjacent white matter on T1-weighted MR images, which is a particular challenge for automated MR image analysis methods (Amini, et al. 2004). Only by com- paring such automated methods with manual investigator- intensive methods can we establish the reliability of volume estimates. The present study provides important data indi- cating the specificity and validity of automated thalamic volume estimation using FreeSurfer software. In particular, further to demonstrating consistency between stereological and FreeSurfer volume estimates of the thalamus in healthy subjects and neurological patients, we demonstrate that the extent of agreeability between stereology and FreeSurfer is equal to the agreeability between two human anatomists estimating thalamic volume using stereological methods. FreeSurfer software is now a frequently used tool for the estimation of subcortical structure volume. At the time of writing, a pubmed search using “Freesurfer” and “volume” yields 87 articles (October 2011). The vast majority of these articles are application studies, particularly in neurological disorders, and only a few have sought to evaluate the valid- ity of volume measurements. Various levels of consistency between FreeSurfer and manual ROI methods have been reported 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), amgydala (Dewey, et al. 2010; Morey, et al. 2009) and striatum (Dewey, et al. 2010). Dewey et al. (2010) performed a series of comparisons between the fully automated techniques of FreeSurfer and Individual Brain Atlases using Statistical Parametric Mapping (IBASPM) with auto-assisted manual tracings of the hippocampus, amygdala, putamen and cau- date. The authors report that FreeSurfer segmentations exhibited significantly higher mean spatial overlap with auto-assisted tracings in all structures compared to IBASPM using dice coefficients. We were not in a position to perform spatial overlap analyses of the thalamus given that stereology and FreeSurfer are two inherently distinct MR image analysis approaches. However, this is one of the primary strengths of the data presented here, insomuch that a reliable volume estimate obtained using a gold-standard (non-voxel labelling) manual approach on MR images with- out automated spatial transformations (i.e. in native space) is comparable to a fully automated approach that requires spatial transformations in order to label an ROI and obtain Neuroinform (2012) 10:341–350 a volume. Our interest was with respect to the reliability of the volume estimate of the thalamus. To our knowledge, the present study is the first to inde- pendently provide data validating the application of FreeSurfer to obtain automated volumes of the left and right thalamus. Based on the congruence between the data obtained from FreeSurfer and manual stereology—the latter of which is considered to represent the ‘gold standard’ approach due to the requirement of an expert anatomist— we recommend the use of FreeSurfer software for accurate volumetric quantification of the thalamus using high- resolution T1-weighted MRI. The removal of an expert Fig. 4 Volume reduction of the left (blue circles) and right (red squares) thalamus in patients with JME relative to healthy controls using stereol- ogy a and FreeSurfer b. Error bars indicate the 95% confidence intervals