The One Click Wonder: a retrained automated segmentation pipeline that enables quantitative and modular analysis of...

High-throughput approaches have transformed the study of gene regulation by enabling quantitative, genome-scale analyses in both genomics and imaging. However, applying these methods to intact organisms remains challenging, particularly for high-throughput, 3D imaging. In Caenorhabditis elegans , generalist segmentation models often perform poorly due to rapid changes in nuclear size, shape, and density. To overcome this obstacle, we developed One Click Wonder (OCW) , an automated pipeline that pairs a retrained Cellpose model with stage-specific parameter selection to deliver accurate, high-throughput segmentation of embryos. We further introduce the Biological Annotation and Association Mapper (BAAM) , which integrates segmentation with spot detection, to enable single-cell quantitation. Applied to the pioneer factor pha-4/FoxA , this pipeline revealed distinct cell populations with an eight-fold range in transcriptional burst frequency. These findings demonstrate that OCW and BAAM provide a modular, scalable pipeline for quantitative, single-cell analysis of gene expression in C. elegans embryos.