Rapid three‐dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development

Abstract

We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three-dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit-scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein-expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales. Developmental Dynamics 235:2940 -2948, 2006.

Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics

“…However, far more information can be extracted from high-speed analysis. Recent advancements in confocal microscopy allow the fast and sensitive imaging of fluorescent blood cells, endocardium, and myocardium when they are in motion; sophisticated computational tools can then reconstruct the optical sections to create four-dimensional depictions of the beating heart [54]. These data sets facilitate calculation of a variety of relevant metrics of cardiac function.…”

“…Reconstruction of the waves of endocardial and myocardial movement and the patterns of blood cell displacement within the contracting heart tube have shown that the fluid dynamics of the heart tube resemble a suction pump, challenging the notion that the heart tube drives circulation via peristalsis (Figure 3) [55]. Examination of temporal changes in chamber volumes, cardiac cushion movements, and patterns of blood flow have documented the maturation of the AV valve into a functional apparatus for preventing backwards flow [54]. This quantitative characterization of the maturation of cardiac function in wild-type embryos provides a powerful framework for the future analysis of mutant phenotypes, although it is important to note that analysis of irregular contractions may require refinement of computational algorithms that are based on assumptions of the rhythmic activity expected in wild-type embryos [54].…”

“…Examination of temporal changes in chamber volumes, cardiac cushion movements, and patterns of blood flow have documented the maturation of the AV valve into a functional apparatus for preventing backwards flow [54]. This quantitative characterization of the maturation of cardiac function in wild-type embryos provides a powerful framework for the future analysis of mutant phenotypes, although it is important to note that analysis of irregular contractions may require refinement of computational algorithms that are based on assumptions of the rhythmic activity expected in wild-type embryos [54].…”

In vivo imaging of the cyclic changes in cross‐sectional shape of the ventricular segment of pulsating embryonic chick hearts at stages 14 to 17: A contribution to the understanding of the ontogenesis of cardiac pumping function

“…According to the traditional view, the cyclic changes in its cross-sectional shape were described as concentric narrowing and widening of a tube of circular cross-section (e.g., Barry, 1948;Liebling et al, 2006). The new in vivo imaging data, however, show that, during the cardiac cycle, only the myocardial tube undergoes almost concentric narrowing and widening while the endocardial tube undergoes eccentric narrowing and widening, having an elliptic cross-section at end-diastole and a slit-shaped crosssection at end-systole (see Fig.…”

3D image stack reconstruction in live cell microscopy of Drosophila muscles and its validation

“…To track leukocytes in the vasculature of mice, a registration method has been proposed that stabilizes jitter caused by circulatory and respiratory movements (16). The 4D reconstruction of the beating heart in zebrafish embryos has been achieved using a wavelet-based synchronization approach (17).…”