The long-term goal of our study is to understand the internal organization of the octocoral stem canals, as well as their physiological and functional role in the growth of the colonies, and finally to assess the influence of climatic changes on this species. Here we focus on imaging tools, namely acquisition and processing of three-dimensional high-resolution images, with emphasis on automated extraction of canal pathways. Our aim was to evaluate the feasibility of the whole process, to point out and solve – if possible – technical problems related to the specimen conditioning, to determine the best acquisition parameters and to develop necessary image-processing algorithms. The pathways extracted are expected to facilitate the structural analysis of the colonies, namely to help observing the distribution, formation and number of canals along the colony. Five volumetric images of Muricea muricata specimens were successfully acquired by X-ray computed tomography with spatial resolution ranging from 4.5 to 25 micrometers. The success mainly depended on specimen immobilization. More than of the canals were successfully detected and tracked by the image-processing method developed. Thus obtained three-dimensional representation of the canal network was generated for the first time without the need of histological or other destructive methods. Several canal patterns were observed. Although most of them were simple, i.e. only followed the main branch or “turned” into a secondary branch, many others bifurcated or fused. A majority of bifurcations were observed at branching points. However, some canals appeared and/or ended anywhere along a branch. At the tip of a branch, all canals fused into a unique chamber. Three-dimensional high-resolution tomographic imaging gives a non-destructive insight to the coral ultrastructure and helps understanding the organization of the canal network. Advanced image-processing techniques greatly reduce human observer's effort and provide methods to both visualize and quantify the structures of interest.