ArticlebioRxiv : the preprint server for biology2026
zFISHer: Automated 3D Registration, Detection, and Colocalization with Interactive Curation for Sequential Multiplexed FISH.
Article in bioRxiv : the preprint server for biology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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3 authors.
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Abstract
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.
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