Three-dimensional fluorescence time-lapse imaging of structural, cellular and subcellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the heart's physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally "freeze" the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heart in vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structures in vivo.

Cardiac injury induces a sustained innate immune response in both zebrafish and mammals. Macrophages, highly plastic immune cells, perform a range of both beneficial and detrimental functions during mammalian cardiac repair yet their precise roles in zebrafish cardiac regeneration are not fully understood. Here we characterise cardiac regeneration in the rapidly regenerating larval zebrafish laser injury model and use macrophage ablation and macrophage-less irf8 mutants to define the requirement of macrophages for key stages of regeneration. We found macrophages to display cellular heterogeneity and plasticity in larval heart injury as in mammals. Live heartbeat-synchronised imaging and RNAseq revealed an early proinflammatory macrophage phase which then resolves to an anti-inflammatory, profibrotic phase. Macrophages are required for cardiomyocyte proliferation but not for functional or structural recovery following injury. Macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium which upregulates mitogen VEGFaa. Experimental perturbation of VEGF signalling confirmed VEGFaa to be an important inducer of cardiomyocyte proliferation revealing a previously unrecognised mechanism by which macrophages aid cardiac regeneration.

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