Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis

Imaging Proteins, Cells, and Tissues Dynamics during Embryogenesis with Two-Photon Light-Sheet Microscopy

Two-photon light sheet microscopy combines nonlinear excitation with the novel sheet-illumination, orthogonal to the detection direction, to achieve high penetration depth, high acquisition speed, and low photodamage, compared with conventional imaging techniques. These advantages allow unprecedented observation of the processes that govern embryogenesis, where the ability to image fast the dynamic three dimensional structure of the developing embryo, over extended periods of time, is critical. We present a selection of applications where two-photon light sheet microscopy is utilized to observe the dynamics of proteins, cells, and tissues, toward an understanding of the construction program of the developing embryos

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture

Light-sheet microscopes must compromise between field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide high resolution but their narrow depth of field fails to capture effectively the fluorescence signal generated by the illumination light sheets, in imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases total fluorescence collection, reduces background, and improves the overall signal-to-noise ratio (SNR). We demonstrate, through numerical simulations and imaging of bead phantoms as well as living animals, that ExD-SPIM increases the SNR by more than three-fold and dramatically reduces the rate of fluorescence photobleaching, when compared to a low-NA system with an equivalent depth of field. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.Comment: 9 pages, 3 figures; supplementary material include

A case of hepatic cyst-induced internal jugular venous thrombosis

• Echocardiography can demonstrate hepatic cyst–induced right atrial compression. • Hepatic cyst–induced blood flow stasis can cause internal jugular venous thrombus. • Laparoscopic deroofing of hepatic cysts is a safe and effective treatment

Dynamic Three-Dimensional Imaging of Cellular Shape Changes and Protein Expression in the Developing Zebrafish Heart

We present our results in dynamic three-dimensional (3D) imaging and quantification of the cellular shape changes and gene expressions of the developing zebrafish heart, in the effort to understand the mechanisms of the embryonic construction of this critical organ. The vertebrate heart is built up through a series of steps taking two flat layers of cells to a hollow heart tube to a multi-layered, multi-chambered, chirally twisted structure of the mature organ. Additionally, the heart is the first organ in the developing embryo to function, through its beating and pumping of the blood, shortly after the formation of the heart tube. Despite this intrinsic dynamic 3D nature of the developing heart, previous works documenting its development consist of largely 2D and/or static imaging (utilizing pharmacological means to stop the beating of the heart), due to the challenges in achieving fast, high 3D-resolution with conventional imaging modalities. To overcome these challenges, we employ 2-photon light sheet microscopy and a wavelet-based synchronization and registration method to achieve the required spatial and temporal resolution to capture the 3D motion of the heart. The high speed 3D imaging and analysis is carried out on several transgenic zebrafish lines that have been recently generated in our lab where proteins important for heart development are fluorescently tagged at their endogenous loci. We thus document not only cellular morphology but also critical genes' expression, with sub-cellular resolution, of the developing heart, over its beating cycle and at different development times. These results provide the necessary groundwork to start deciphering the process where the dynamic changes in cellular shapes, gene expressions, and cellular physical properties participate, in concert with the genetic program, in the development of the vertebrate heart

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines

Imaging the Beating Heart with Macroscopic Phase Stamping

We present a novel approach for imaging the beating embryonic heart, based on combining two independent imaging channels to capture the full spatio-temporal information of the moving 3D structure. High-resolution, optically-sectioned image recording is accompanied by simultaneous acquisition of low-resolution, whole-heart recording, allowing the latter to be used in post-acquisition processing to determine the macroscopic spatio-temporal phase of the heart beating cycle. Once determined, or 'stamped', the phase information common to both imaging channels is used to reconstruct the 3D beating heart. We demonstrated our approach in imaging the beating heart of the zebrafish embryo, capturing the entire heart over its full beating cycle, and characterizing cellular dynamic behavior with sub-cellular resolution

Live 4D Imaging of the Embryonic Vertebrate Heart with Two-Photon Light Sheet Microscopy and Simultaneous Optical Phase Stamping

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