Di Sante and colleagues present CALIPERS, a framework that makes cell-cycle state a measurable variable in live-cell imaging, pairing a redesigned reporter with automated inference across human cells,...

Di Sante and colleagues present CALIPERS, a framework that makes cell-cycle state a measurable variable in live-cell imaging, pairing a redesigned reporter with automated inference across human cells,...

$${\rm{Micro}}{\mathbb{S}}{\rm{plit}}$$ M i c r o S p l i t is a computational unmixing method for multiplexed fluorescence imaging of up to four structures per imaging channel.

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Cell cycle progression, migration, and proliferation shape development and regeneration, but simultaneous live-cell imaging remains challenging as conventional fluorescent cell cycle indicators (FUCCI) monopolize the green and red channels used by most structural and functional biosensors. To overcome this, we integrated a spectrally re-engineered FUCCI variant, open-source analysis software, and four-color human stem cell reporter lines into CALIPERS: a method for Cell-cycle-Aware Live-cell Imaging in Phenotyping and Regeneration Studies. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 852560 European Union, https://ror.org/019w4f821, 101140192 Italian Ministry of Enterprises and Made in Italy, F13C23003260003

An optical pooled cell profiling platform (PERISCOPE) based on Cell Painting and optical sequencing of molecular barcodes was used to develop the first unbiased genome-wide morphology-based perturbati...