Image analysis pipeline for segmentation of a biological porosity network, the lacuno-canalicular system in stingray tesserae.

A prerequisite for many analysis tasks in modern comparative biology is the segmentation of 3-dimensional (3D) images of the specimens being investigated (e.g. from microCT data). Depending on the specific imaging technique that was used to acquire the images and on the image resolution, different segmentation tools are required. While some standard tools exist that can often be applied for specific subtasks, building whole processing pipelines solely from standard tools is often difficult. Some tasks may even necessitate the implementation of manual interaction tools to achieve a quality that is sufficient for subsequent analysis. In this work, we present a pipeline of segmentation tools that can be used for the semiautomatic segmentation and quantitative analysis of voids in tissue (i.e. internal structural porosity). We use this pipeline to analyze lacuno-canalicular networks in stingray tesserae from 3D images acquired with synchrotron microCT.•The first step of this pipeline, the segmentation of the tesserae, was performed using standard marker-based watershed segmentation.•The efficient processing of the next two steps, that is, the segmentation of all lacunae spaces belonging to a specific tessera and the separation of these spaces into individual lacunae required recently developed, novel tools.•For error correction, we developed an interactive method that allowed us to quickly split lacunae that were accidentally merged, and to merge lacunae that were wrongly split.•Finally, the tesserae and their corresponding lacunae were subdivided into structural wedges (i.e. specific anatomical regions) using a semi-manual approach. With this processing pipeline, analysis of a variety of interconnected structural networks (e.g. vascular or lacuno-canalicular networks) can be achieved in a comparatively high-throughput fashion. In our study system, we were able to efficiently segment more than 12,000 lacunae in high-resolution scans of nine tesserae, providing a robust data set for statistical analysis.