Functional neuroimaging of small brainstem structures in humans is gaining increasing interest due to their potential importance in aging and many clinical conditions. Researchers have used different methods to measure activity in the locus coeruleus (LC), the main noradrenergic nucleus in the brain. However, the reliability of the different methods for identifying this small structure is unclear. In the present article, we compared four different approaches to estimate localization of the LC in a large sample (N = 98): 1) a probabilistic map from a previous study, 2) masks segmented from neuromelanin-sensitive scans, 3) components from a masked-independent components analysis of the functional data, and 4) a mask from pupil regression of the functional data. The four methods have been used in the community and find some support as reliable ways of assessing the localization of LC in vivo in humans by using functional imaging. We report several measures of similarity between the LC masks obtained from the different methods. In addition, we compare the similarity between functional connectivity maps obtained from the different masks. We conclude that sample-specific masks appear more suitable than masks from a different sample, that masks based on structural versus functional methods may capture different portions of LC, and that, at the group level, the creation of a "consensus" mask using more than one approach may give a better estimate of LC localization. ### Competing Interest Statement The authors have declared no competing interest.
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