A multimodal perturbation atlas defines the phenotypic resolution of cellular morphology.

Because cells are complex dynamical systems, modeling cellular behaviors requires methods that capture how cells evolve across time, environments, and interventions. Microscopy is uniquely suited to this goal in that it can be applied to living cells in their native context. However, the phenotypic resolving power of live-cell microscopy remains incompletely characterized, particularly relative to molecular assays. Here, we present a multimodal perturbation atlas of 1,000 pooled CRISPR knockouts in A549 cells, profiled by fluorescence microscopy (39 live, 13 fixed markers), label-free phase imaging of the same live cells, and single-cell RNA sequencing (scRNA-seq). Totaling ~57 million single-cell profiles, our data yield rich cell-biological signatures that map individual gene function. We find that phase imaging matches -- and, with sufficient cell coverage, exceeds -- the phenotypic resolution of fluorescence imaging and scRNA-seq, while capturing higher-order pathway organization that scRNA-seq does not resolve. These results establish intrinsic morphology as a high-precision readout of cellular state, and lay a foundation for live-cell profiling of phenotypic trajectories.