Tapenade: Spatial Quantification of Mechanical and Genetic Fields in Dense 3D Organoids From Cell to Tissue Scale

Whole-mount 3D imaging of multilayered biological tissues enables quantitative analysis of cell states and organization in their spatial context. However, extracting unbiased and meaningful quantitative information from dense, multilayered samples remains challenging due to imaging artifacts, increased density, and limited signal-to-noise ratio. Open source bioimage analysis workflows tailored to this type of analysis are scarce, and analysis bottlenecks like image curation or cell segmentation are seldom available without coding expertise. Here, we present a step-by-step computational protocol for the analysis of dense 3D organoid datasets using the Tapenade (Thorough Analysis PipEliNe for Advanced DEep imaging) workflow. Starting from multichannel image stacks, the protocol guides users through software installation, registration and fusion of multi-view datasets, preprocessing, and nuclei segmentation. It further details the generation of quantitative outputs, including morphometric measurements, deformation fields, and spatial correlation analyses. The workflow can be executed through open-source Python scripts or user-friendly Napari interfaces, allowing interactive parameter tuning and 3D visualization at each stage. This pipeline provides an accessible and modular framework for nonspecialist users to perform reproducible, multiscale quantitative analysis of 3D organoid images, while retaining flexibility for advanced users to customize individual steps. Key features • Multiscale quantitative analysis of dense 3D tissues: developed on thick whole-mount immunostained stem cell aggregates, Tapenade enables quantitative analysis from the cellular to the tissue scale. • End-to-end pipeline: Tapenade provides graphical interfaces, notebooks, and step-by-step tutorials to streamline preprocessing and quantitative measurements, including morphometrics, deformation fields, and spatial correlations. • Integrated visualization and modular analysis: Napari plugins support interactive parameter tuning and real-time exploration of large volumetric datasets. • Scalability: Tapenade is compatible with standard bioimaging formats (e.g., TIFF, OME-TIFF, CZI, Zarr) and scalable to large 3D datasets through lazy loading and batch-processing strategies.