Neuroinform (2012) 10:341–350 Table 1 Inter-rater intra-class coefficients for volumetric measures Rater 1 Rater 2 Left thalamus Rater 1 Rater 2 FS Right thalamus Rater 1 Rater 2 FS − 0.977 0.969 − 0.952 0.923 0.977 − 0.925 0.952 − 0.916 FS 0.969 0.925 − 0.923 0.916 − 345 approaches are presented in Table 1. Measurements between R1 and R2, and between manual raters and FreeSurfer, achieve high intra-class correlations (all >0.9). Figure 3 presents the relationship between stereological (R1) and FreeSurfer estimates of left and right thalamus volume across the entire sample of 72 subjects investigated in the present study. It is immediately obvious that the two methods yield consistent thalamic volumes. Intra-class corre- lations revealed a slightly reduced level of consistency be- tween human and FreeSurfer analysis of the entire sample relative to the sub-sample of ten subjects (left thalamus 0 0.812, right thalamus0 0.881). Mean (SD) left and right tha- lamic volume was 7422.4 mm3 (824.3) and 7390.1 mm3 (805.8) for stereology and 7343.6 mm3 (824.3) and 7388.3 mm3 (844.0) for FreeSurfer. There was no difference between the volume of the left and right thalamus across all 72 subjects using stereology (F 0 0.09, p 0 0.82) or FreeSurfer (F 0 0.10, p 0 0.75) (and no differences when patients and controls were separated, p>0.80). Although there were occa- sional differences in the direction of inter-hemispheric thalam- ic volume asymmetry in individual cases between stereology and FreeSurfer (Fig. 3, right panel), this did not represent a statistically significant group effect (F 0 1.46, p0 0.23). B. Stereology vs Freesurfer: Identification of thalamic at- rophy in JME Both techniques were equally sensitive in detecting bilateral thalamic volume atrophy in patients with JME relative to controls (Fig. 4). Using stereology, mean (SD) left and right thalamic volume was 6843.2 mm3 (746.6) and 6763.3 mm3 (824.0) in patients with JME, and 7507.8 mm3 (805.6) and 7482.6 mm3 (767.2) in controls, respectively. Volume reduc- tion in patients was found to be statistically significant for the left (F(1,70)0 5.43, p0 0.02) and right (F(1,70)0 6.77, p0 0.01) thalamus compared to controls. Using FreeSurfer, mean (SD) left and right thalamic volume was 6803.4 mm3 (732.8) and 6750.0 mm3 (714.3) in patients with JME, and 7430.7 mm3 (809.9) and 7491.3 mm3 (822.3) in controls, respectively. controls (n 0 72). Univariate ANOVAs were used to investi- gate patient-control differences in volumes, and corrected for multiple comparisons using Statistica version 9.1, (Stat Soft. Inc, www.statsoft.com). Results A. Stereology vs FreeSurfer: Consistency between volu- metric measures Figure 2 shows the comparison of the three approaches (rater one (R1) for stereology, rater two (R2) for stereology and FreeSurfer) to estimate thalamic volume in the randomly selected ten subjects. This inter-rater / between-technique analysis indicates consistency across measures, and most no- tably, that FreeSurfer performed at least as consistent as R2 relative to R1. Mean (SD) left and right thalamic volume was 7339.6 mm3 (567.3) and 7339.2 mm3 (489.3) for R1, 7456.3 mm3 (597.3) and 7444.3 mm3 (590.6) for R2, and 7365.0 mm3 (641.3) and 7317.6 mm3 (610.0) for FreeSurfer, respectively. Intra-class correlations across raters and Fig. 3 Relationship between stereological and FreeSurfer volume esti- mates of the left a and right b thalamus in the whole study sample. The relationship between left-right asymmetries determined for each individ- ual participant by each technique is shown in c. Although stereology and FreeSurfer determined left-right asymmetries of the thalamus in the same direction for the vast majority of subjects (lower left and upper right quandrants), there were some disassociations (top left quadrant)