AI-assisted quantification of the clonogenic assay

The clonogenic assay is the foundational standard for evaluating cellular radiosensitivity, yet its manual quantification is limited by labor-intensive processing, high inter-observer variability, and a reductive binary scoring system. Here, we present and validate a deep learning-based segmentation pipeline for the automated, high-throughput quantification of mammalian cell survival across diverse radiation qualities (photons, protons, and carbon ions). By benchmarking against expert consensus using Bland-Altman analysis, we demonstrate that this AI framework consistently operates within natural human error margins. Crucially, transitioning to automated whole-well image-based analysis enables the extraction of continuous multidimensional data, including colony area, density, and cellularity. This phenotypic profiling successfully identified and quantified high-cellularity colonies formed by radioresistant BxPC-3 cells under particle irradiation. Coupled with rapid image acquisition platforms, this methodology significantly reduces processing bottlenecks while standardizing the derivation of relative biological effectiveness (RBE). By advancing beyond binary survival metrics to continuous morphological profiling, this framework provides a vital new analytical dimension for investigating structural mechanisms of radioresistance.