Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences

Being able to acquire, visualize, and analyze 3D time series (4D data) from living embryos makes it possible to understand complex dynamic movements at early stages of embryonic development. Despite recent technological breakthroughs in 2D dynamic imaging, confocal microscopes remain quite slow at capturing optical sections at successive depths. However, when the studied motion is periodic— such as for a beating heart—a way to circumvent this problem is to acquire, successively, sets of 2D+time slice sequences at increasing depths over at least one time period and later rearrange them to recover a 3D+time sequence. In other imaging modalities at macroscopic scales, external gating signals, e.g., an electro-cardiogram, have been used to achieve proper synchronization. Since gating signals are either unavailable or cumbersome to acquire in microscopic organisms, we have developed a procedure to reconstruct volumes based solely on the information contained in the image sequences. The central part of the algorithm is a least-squares minimization of an objective criterion that depends on the similarity between the data from neighboring depths. Owing to a wavelet-based multiresolution approach, our method is robust to common confocal microscopy artifacts. We validate the procedure on both simulated data and in vivo measurements from living zebrafish embryos

Nonuniform temporal alignment of slice sequences for four-dimensional imaging of cyclically deforming embryonic structures

The temporal alignment of nongated slice-sequences acquired at different axial positions in the living embryonic zebrafish heart permits the reconstruction of dynamic, three-dimensional data. This approach overcomes the current acquisition- speed limitation of confocal microscopes for real-time three-dimensional imaging of fast processes. Current synchronization methods align and uniformly scale the data in time, but do not compensate for slight variations in the heart rhythm that occur within a heartbeat. Therefore, they impose constraints on the admissible data quality. Here, we derive a nonuniform registration procedure based on the minimization of the absolute value of the intensity difference between adjacent slice-sequence pairs. The method compensates for temporal intra-sample variations and allows the processing of a wider range of data to build functional, dynamic models of the beating embryonic heart. We show reconstructions from data acquired in living, fluorescent zebrafish embryos

Quantitative mapping of intracardiac blood flow in embryonic zebrafish

Using real-time in vivo imaging and digital particle image velocimetry (DPIV) we quantitatively described the intracardiac flow environment of early zebrafish (Danio rerio) embryos. Gross cardiac dynamics were defined for two embryonic stages: 4.5 days post fertilization (dpf) and 37 hours post fertilization (hpf) using high-speed transmitted light microscopy with valve dynamics visualized through high-speed laser-scanning microscopy on transgenic embryos expressing GFP

The Embryonic Vertebrate Heart Tube Is a Dynamic Suction Pump

The embryonic vertebrate heart begins pumping blood long before the development of discernable chambers and valves. At these early stages, the heart tube has been described as a peristaltic pump. Recent advances in confocal laser scanning microscopy and four-dimensional visualization have warranted another look at early cardiac structure and function. We examined the movement of cells in the embryonic zebrafish heart tube and the flow of blood through the heart and obtained results that contradict peristalsis as a pumping mechanism in the embryonic heart. We propose a more likely explanation of early cardiac dynamics in which the pumping action results from suction due to elastic wave propagation in the heart tube

Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis

Wavelet-based Synchronization of Nongated Confocal Microscopy Data for 4D Imaging of the Embryonic Heart Invited Paper

With the availability of new confocal laser scanning microscopes, fast biological processes, such as the blood flow in living organisms at early stages of the embryonic development, can be observed with unprecedented time resolution. When the object under study has a periodic motion, e.g. a beating embryonic heart, the imaging capabilities can be extended to retrieve 4D data. We acquire nongated slice-sequences at increasing depth and retrospectively synchronize them to build dynamic 3D volumes. Here, we present a synchronization procedure based on the temporal correlation of wavelet features. The method is designed to handle large data sets and to minimize the influence of artifacts that are frequent in fluorescence imaging techniques such as bleaching, nonuniform contrast, and photon-related noise

Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis

The pattern of blood flow in the developing heart has long been proposed to play a significant role in cardiac morphogenesis. In response to flow-induced forces, cultured cardiac endothelial cells rearrange their cytoskeletal structure and change their gene expression profiles. To link such in vitro data to the intact heart, we performed quantitative in vivo analyses of intracardiac flow forces in zebrafish embryos. Using in vivo imaging, here we show the presence of high-shear, vortical flow at two key stages in the developing heart, and predict flow-induced forces much greater than might have been expected for micro-scale structures at low Reynolds numbers. To test the relevance of these shear forces in vivo, flow was occluded at either the cardiac inflow or outflow tracts, resulting in hearts with an abnormal third chamber, diminished looping and impaired valve formation. The similarity of these defects to those observed in some congenital heart diseases argues for the importance of intracardiac haemodynamics as a key epigenetic factor in embryonic cardiogenesis

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

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