These files enable automated image processing related to the enGLOW publication, which details the enteric network Gastrointestinal Light-sheet Optical Workflow. This workflow produces high-quality 3D light-sheet images of animal and human gastrointestinal samples of multiple centimeters in size. These algorithms can be used to: virtually flatten 1- or 2-channel images to separate layers of the gut wall, plot extracted XYZ coordinates of segmented cells in 3D and unwrap XYZ coordinates from 3D datasets to be plotted in 2D. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 853378).