Michael Liebling

Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart.

The directionality of local blood flow in the zebrafish embryonic heart is essential for proper heart valve formation.

Fresnelets: new multiresolution wavelet bases for digital holography

We propose a construction of new wavelet-like bases that are well suited for the reconstruction and processing of optically generated Fresnel holograms recorded on CCD-arrays. The starting point is a wavelet basis of L2 to which we apply a unitary Fresnel transform. The transformed basis functions are shift-invariant on a level-by-level basis but their multiresolution properties are governed by the special form that the dilation operator takes in the Fresnel domain. We derive a Heisenberg-like uncertainty relation that relates the localization of Fresnelets with that of their associated wavelet basis. According to this criterion, the optimal functions for digital hologram processing turn out to be Gabor functions, bringing together two separate aspects of the holography inventors work. We give the explicit expression of orthogonal and semi-orthogonal Fresnelet bases corresponding to polynomial spline wavelets. This special choice of Fresnelets is motivated by their near-optimal localization properties and their approximation characteristics. We then present an efficient multiresolution Fresnel transform algorithm, the Fresnelet transform. This algorithm allows for the reconstruction (backpropagation) of complex scalar waves at several user-defined, wavelength-independent resolutions. Furthermore, when reconstructing numerical holograms, the subband decomposition of the Fresnelet transform naturally separates the image to reconstruct from the unwanted zero-order and twin image terms. This greatly facilitates their suppression. We show results of experiments carried out on both synthetic (simulated) data sets as well as on digitally acquired holograms.

Autofocus for digital Fresnel holograms by use of a Fresnelet-sparsity criterion

We propose a robust autofocus method for reconstructing digital Fresnel holograms. The numerical reconstruction involves simulating the propagation of a complex wave front to the appropriate distance. Since the latter value is difficult to determine manually, it is desirable to rely on an automatic procedure for finding the optimal distance to achieve high-quality reconstructions. Our algorithm maximizes a sharpness metric related to the sparsity of the signals expansion in distance-dependent waveletlike Fresnelet bases. We show results from simulations and experimental situations that confirm its applicability.

Complex-Wave Retrieval from a Single Off-Axis Hologram

We present a new digital two-step reconstruction method for off-axis holograms recorded on a CCD camera. First, we retrieve the complex object wave in the acquisition plane from the holograms samples. In a second step, if required, we propagate the wave front by using a digital Fresnel transform to achieve proper focus. This algorithm is sufficiently general to be applied to sophisticated optical setups that include a microscope objective. We characterize and evaluate the algorithm by using simulated data sets and demonstrate its applicability to real-world experimental conditions by reconstructing optically acquired holograms.

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.

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. Developmental Dynamics 235:2940–2948, 2006.

Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development

VIDEO ABSTRACT The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

Stereotypical Cell Division Orientation Controls Neural Rod Midline Formation in Zebrafish

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body-axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Here we show that SDO is required for formation of the neural rod midline during neurulation but dispensable for elongation of the body axis during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the noncanonical Wnt receptor Frizzled 7 (Fz7) and that interfering with cell division orientation leads to severe defects in neural rod midline formation but not body-axis elongation. These findings suggest a novel function for Fz7-controlled cell division orientation in neural rod midline formation during neurulation.

Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real‐time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever‐faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post‐processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field.

High-resolution imaging of cardiomyocyte behavior reveals two distinct steps in ventricular trabeculation

Over the course of development, the vertebrate heart undergoes a series of complex morphogenetic processes that transforms it from a simple myocardial epithelium to the complex 3D structure required for its function. One of these processes leads to the formation of trabeculae to optimize the internal structure of the ventricle for efficient conduction and contraction. Despite the important role of trabeculae in the development and physiology of the heart, little is known about their mechanism of formation. Using 3D time-lapse imaging of beating zebrafish hearts, we observed that the initiation of cardiac trabeculation can be divided into two processes. Before any myocardial cell bodies have entered the trabecular layer, cardiomyocytes extend protrusions that invade luminally along neighboring cell-cell junctions. These protrusions can interact within the trabecular layer to form new cell-cell contacts. Subsequently, cardiomyocytes constrict their abluminal surface, moving their cell bodies into the trabecular layer while elaborating more protrusions. We also examined the formation of these protrusions in trabeculation-deficient animals, including erbb2 mutants, tnnt2a morphants, which lack cardiac contractions and flow, and myh6 morphants, which lack atrial contraction and exhibit reduced flow. We found that, compared with cardiomyocytes in wild-type hearts, those in erbb2 mutants were less likely to form protrusions, those in tnnt2a morphants formed less stable protrusions, and those in myh6 morphants extended fewer protrusions per cell. Thus, through detailed 4D imaging of beating hearts, we have identified novel cellular behaviors underlying cardiac trabeculation.