A road map to manual segmentation of cerebral structures

Manual segmentation is an essential tool in the researcher's technical arsenal. It is a frequent practice necessary for image analysis in many protocols, especially in neuroimaging and comparative brain anatomy. In the framework of emergence of studies focusing on alternative animal models, manual segmentation procedures play a critical role. Nevertheless, this critical task is often assigned to students, a process that, unfortunately, tends to be time-consuming and repetitive. Well-conducted and well-described segmentation procedures can potentially guide novice and even expert operators and enhance research works' internal and external validity, making it possible to harmonize studies and facilitate data sharing. Furthermore, recent advances in neuroimaging, such as ex vivo imaging or ultra-high-field MRI, enable new acquisition modalities and the identification of minute structures that are barely visible with typical approaches. In this context of increasingly detailed and multimodal brain studies, reflecting on methodology is relevant and necessary. Because it is crucial to implement good practices in manual segmentation per se but also in the description of the segmentation procedures in research papers, we propose a general roadmap for optimizing the technique, its process and the reporting of manual segmentation. For each of them, the relevant elements of the literature have been collected and cited. The article is accompanied by a checklist that the reader can use to verify that the critical steps are being followed.