The developing vertebrate heart is a highly dynamic organ that starts to function early on during embryonic development, even as it continues to undergo dramatic morphological changes and cellular differentiation. Fast and high resolution three-dimensional (3D) imaging is needed to document the intrinsic cellular dynamics of the beating heart, as a critical step in understanding its development. To meet the challenges of obtaining sub-cellular resolution imaging of a dynamic 100-micron length scale 3D structure, which moves quasi-periodically at frequency of a few Hertz, over tens of microns amplitude, we have employed two-photon light sheet microscopy (2p-SPIM) and a novel independent optical phase stamping method to generate well-resolved 3D movies (4D) of the beating heart. Applying this 4D imaging modality to zebrafish embryos, we have found remarkable heterogeneity in cardiomyocyte morphology, gene expression, and behavior both during the cardiac cycle, and over the developmental time. The observed heterogeneity appears to play a key role in the maintenance of tissue geometry and cardiac output as the heart undergoes cycles of contraction and expansion. The variation in cellular morphology and behavior provide new insights into the tight link between cellular dynamics, mechanical environment exerted and felt by the beating heart, and the genetic program that governs not only the differentiation and construction but also the maintenance of this important organ.
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