zFISHer: Automated 3D Registration, Detection, and Colocalization with Interactive Curation for Sequential Multiplexed FISH
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Summary
Sequential multiplexed fluorescence in situ hybridization (FISH) enables spatially resolved molecular profiling in cell monolayers, but analyzing puncta colocalization across three-dimensional (3D) datasets remains a labor-intensive bottleneck. zFISHer is an open-source application built on the napari viewer that provides complete automation of sequential FISH image processing in conjunction with interactive user-curation tools. zFISHer provides end-to-end analysis of paired FISH datasets, encompassing nuclear segmentation, automated puncta detection on unaligned z-stacks, multi-round image registration via translation-constrained RANSAC with optional B-spline deformable warping, precise transformation of puncta coordinates into aligned space, consensus nuclei generation, interactive editing with real-time collision detection, and pairwise and tri-channel colocalization analysis with statistics. This includes a “Fishing Hook” raycasting algorithm that enables users to locate puncta at their true 3D centroids by identifying intensity maxima along the camera ray, eliminating manual z-slice navigation, complemented by a sub-voxel volume optimization. The included batch processing mode enables high-throughput unattended analysis of multiple experimental datasets.
Availability and Implementation
zFISHer is open source under the MIT license, freely available on GitHub: https://github.com/stjude/zFISHer . The example dataset (deconvolved ND2 image stacks) is archived on Zenodo at https://doi.org/10.5281/zenodo.20288536 . zFISHer is developed in Python utilizing the napari viewer for the interface. Documentation and expected test outputs for the sample dataset are available on the GitHub: https://github.com/stjude/zFISHer . To report an issue using zFISHer or contributing to it, please file an issue in the GitHub repository: https://github.com/stjude/zFISHer/issues .
Contact
Seth.Staller@STJUDE.ORG
Supplementary Information
Supplementary data are available online.
