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bioRxiv · 10.64898/2026.05.19.726314

zFISHer: Automated 3D Registration, Detection, and Colocalization with Interactive Curation for Sequential Multiplexed FISH

Staller, S. A.·Valentine, V.·Burden, S.
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Abstract

SummarySequential 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 ImplementationzFISHer 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. ContactSeth.Staller@STJUDE.ORG Supplementary InformationSupplementary data are available online.

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Staller, S. A., Valentine, V., Burden, S.. 2026-05-21. zFISHer: Automated 3D Registration, Detection, and Colocalization with Interactive Curation for Sequential Multiplexed FISH. https://doi.org/10.64898/2026.05.19.726314

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