Development and regeneration are shaped by cell cycle progression, migration, and proliferation, but simultaneous live-cell imaging is still difficult since most structural and functional biosensors only use the green and red channels of conventional fluorescent cell cycle indicators (FUCCI). 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.  This novel approach will extend to live-cell imaging the same advantages that cell-cycle awareness has already brought to –omics disciplines.

Deploying CALIPERS in HaCaT cells and hiPSC-derived cardiomyocytes
Live imaging of HaCaT cells expressing FUCCIplex (cyan/magenta) and RFP-LifeAct (grey). To showcase CALIPERS, FUCCIplex is coexpressed in HaCaT cells with  the actin-binding peptide RFP-LifeAct and used live-cell fluorescence imaging over 40 hours to track CC phase duration. Practically, the nucleus of FUCCIplex cells contains CFP in G1, both CFP and iRFP in the G1-S transition, and only iRFP during the S/G2/M phases.
Representative images and structural and functional analyses of hiPSC-CMs re-entering the CC (iRFP-expressing nuclei) for proliferation, multinucleation, or endoreplication. Dashed squares and arrowheads indicate a cell pre and post event in all conditions; the asterisk indicates an additional proliferation event in the endoreplication panels.  Scale bars, 25 μm.