AbstractThree-dimensional fluorescence time-lapse imaging of the beating heart is extremely challenging, due to the heart’s constant motion and a need to avoid pharmacological or phototoxic damage. Although real-time triggered imaging can computationally “freeze” the heart for 3D imaging, no previous algorithm has been able to maintain phase-lock across developmental timescales. We report a new algorithm capable of maintaining day-long phase-lock, permitting routine acquisition of synchronised 3D + time video time-lapse datasets of the beating zebrafish heart. This approach has enabled us for the first time to directly observe detailed developmental and cellular processes in the beating heart, revealing the dynamics of the immune response to injury and witnessing intriguing proliferative events that challenge the established literature on cardiac trabeculation. Our approach opens up exciting new opportunities for direct time-lapse imaging studies over a 24-hour time course, to understand the cellular mechanisms underlying cardiac development, repair and regeneration.