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

Probing cilia-driven flow in living embryos using femtosecond laser ablation and fast imaging

Embryonic development strictly depends on fluid dynamics. As a consequence, understanding biological fluid dynamic is essential since it is unclear how flow affects development. For example, the specification of the left-right axis in vertebrates depends on fluid flow where beating cilia generate a directional flow necessary for breaking the embryonic symmetry in the so-called left-right organizer. To investigate flow dynamics in vivo proper labeling methods necessitate approaches that are compatible with both normal biology and in vivo imaging. In this study, we describe a strategy for labeling and analyzing microscopic fluid flows in vivo that meets this challenge. We developed an all-optical approach based on three steps. First we used sub-cellular femtosecond laser ablation to generate fluorescent micro-debris to label the flow. The non-linear effect used in this technique allows a high spatial confinement and a low invasiveness, thus permitting the targeting of sub-cellular regions deep inside the embryo. Then, we used fast confocal imaging and 3Dparticle tracking were used to image and quantify the seeded flow. This approach was used to investigate the flow generated within zebrafish left-right organizer, a micrometer scale ciliated vesicle located deep inside the embryo and involved in breaking left-right embryonic symmetry. We mapped the velocity field within the vesicle and surrounding a single beating cilium, and showed that this method can address the dynamics of cilia-driven flows at multiple length scales. We could validate the flow features as predicted from previous simulations. Such detailed descriptions of fluid movements will be valuable in unraveling the relationships between cilia-driven flow and signal transduction. More generally, this all-optical approach opens new opportunities for investigating microscopic flow in living tissues

Quantitative imaging of the collective cell movements shaping an embryo

The recent development of imaging and image processing techniques, such as 4D microscopy and 3D cell tracking, enables analysis through quantification of the movement of large cell populations in vivo. These imaging approaches provide an opportunity to study embryonic morphogenesis during development from the level of cellular processes to the scale of entire organism. Image analysis reveals cell collective behaviors that shape an embryo and offers some surprising insights into the cell-cell interactions involved in concerted movements. We illustrate the power of this approach by studying the early development of Drosophila embryos

Structure sensitivity in third-harmonic generation microscopy

International audienceWe characterize experimentally the influence of sample structure and beam focusing on signal level in third-harmonic generation (THG) microscopy. In the case of a homogeneous spherical sample, the dependence of the signal on the size of the sphere can be controlled by modifying the Rayleigh length of the excitation beam. More generally, the influence of excitation focusing on the signal depends on sample geometry, allowing one to highlight certain structures within a complex system. We illustrate this point by focusing-based contrast modulation in THG images of Drosophila embryos

Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration

An all-optical approach for probing microscopic flows in living embryos

Living systems rely on fluid dynamics from embryonic development to adulthood. To visualize biological fluid flow, devising the proper labeling method compatible with both normal biology and in vivo imaging remains a major experimental challenge. Here, we describe a simple strategy for probing microscopic fluid flows in vivo that meets this challenge. An all-optical procedure combining femtosecond laser ablation, fast confocal microscopy and 3D-particle tracking was devised to label, image and quantify the flow. This approach is illustrated by studying the flow generated within a micrometer scale ciliated vesicle located deep inside the zebrafish embryo and involved in breaking left-right embryonic symmetry. By mapping the velocity field within the vesicle and surrounding a single beating cilium, we show this method can address the dynamics of cilia-driven flows at multiple length scales, and can validate the flow features as predicted from previous simulations. This approach provides new experimental access to questions of microscopic fluid dynamics in vivo

Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration

The concerted movement of cells from different germ layers contributes to morphogenesis during early embryonic development. Using an optimized imaging approach and quantitative methods, we analyzed the trajectories of hundreds of ectodermal cells and internalized mesodermal cells within Drosophila embryos over 2 hours during gastrulation. We found a high level of cellular organization, with mesoderm cell movements correlating with some but not all ectoderm movements. During migration, the mesoderm population underwent two ordered waves of cell division and synchronous cell intercalation, and cells at the leading edge stably maintained position. Fibroblast growth factor (FGF) signaling guides mesodermal cell migration; however, we found some directed dorsal migration in an FGF receptor mutant, which suggests that additional signals are involved. Thus, decomposing complex cellular movements can provide detailed insights into collective cell migration

Multicolor two-photon light-sheet microscopy

International audienceTwo-photon microscopy is the most effective approach for deep-tissue fluorescence cellular imaging; however, its application to high-throughput or high-content imaging is often hampered by low pixel rates, challenging multicolor excitation and potential cumulative photodamage. To overcome these limitations, we extended our prior work and combined two-photon scanned light-sheet..

Microscopies multiharmoniques pour l'imagerie structurale de tissus intacts [Second- and third-harmonic generation microscopies for the structural imaging of intact tissues]

International audienceDepuis son introduction en 1990, la microscopie de fluorescence excitée à deux photons (Fluo-2P) s'est peu à peu imposée comme une méthode incontournable d'imagerie de tissus intacts à l'échelle sub-cellulaire. En effet, la caractéristique la plus remarquable de la microscopie multiphotonique est de maintenir une résolution tridimensionnelle micrométrique lors de l'observation en profondeur d'un milieu optiquement diffusant. Combinée aux technologies de protéines-fusion (type GFP), cette approche est aujourd'hui utilisée dans de nombreux domaines, notamment en neurophysiologie. Un autre attrait de ce type d'imagerie réside dans l'utilisation possible d'autres phénomènes optiques non linéaires (c'est-à-dire impliquant l'interaction simultanée de plusieurs photons avec une molécule observée) comme source de contraste. Ainsi, les microscopies par génération de second harmonique (GSH) et par génération de troisième harmonique (GTH) permettent également d'observer des milieux complexes et fournissent des informations complémentaires par rapport à l'imagerie de fluorescence. Certaines structures cellulaires ou tissulaires fournissent, en effet, ce type de réponse optique sans nécessiter de marquage exogène. La microscopie GSH permet, par exemple, de détecter le collagène fibrillaire et la microscopie GTH permet d'observer sans marquage le développement embryonnaire de petits organismes. One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies. In recent years, significant progress has been made in understanding the contrast mechanisms of these recent methodologies, and high-resolution imaging based on intrinsic sources of signal has been demonstrated in cells and tissues. Harmonic generation exhibits structural rather than chemical specificity and can be obtained from a variety of non-fluorescent samples. SHG is observed specifically in dense, non-centrosymmetric arrangements of polarizable molecules, such as collagen fibrils, myofilaments, and polarized microtubule bundles. SHG imaging is therefore emerging as a novel approach for studying processes such as the physiopathological remodelling of the collagen matrix and myofibrillogenesis in intact tissue. THG does not require a non-centrosymmetric system; however no signal can be obtained from a homogeneous medium. THG imaging therefore provides maps of sub-micrometer heterogeneities (interfaces, inclusions) in unstained samples, and can be used as a general purpose structural imaging tool. Recent studies showed that this technique can be used to image embryo development in small organisms and to characterize the accumulation of large lipid bodies in specialized cells. SHG and THG microscopy both rely on femtosecond laser technology and are easily combined with two-photon microscopy

Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos

International audienceWe demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos