Immobilisation of living coral embryos and larvae.

Embedding and immobilisation of living cells and microorganisms is used in a variety of research and commercial applications. Here we report the successful extended immobilisation of coral larvae in a low-gelling temperature agarose. Embryos and larvae of five broadcast-spawning Scleractinian species were immobilised in agarose gel and tested in a series of exploratory survival and settlement assays. The optimal developmental stage for immobilisation was after ciliation at approximately 24 hours post-fertilisation, after which, survival of immobilised larvae of all species was nearly 100%. In long-term assays, 50% of Montipora digitata larvae survived immobilised for 89 days. Furthermore, immobilised larvae of multiple species, that were released from the agarose, generally remained capable of settlement. These results demonstrate that the immobilisation of the early life-history stages of corals is possible for a variety of applications in basic and applied science.

Fluorescence techniques in developmental biology.

Advanced fluorescence techniques, commonly known as the F-techniques, measure the kinetics and the interactions of biomolecules with high sensitivity and spatiotemporal resolution. Applications of the F-techniques, which were initially limited to cells, were further extended to study in vivo protein organization and dynamics in whole organisms. The integration of F-techniques with multi-photon microscopy and light-sheet microscopy widened their applications in the field of developmental biology. It became possible to penetrate the thick tissues of living organisms and obtain good signal-to-noise ratio with reduced photo-induced toxicity. In this review, we discuss the principle and the applications of the three most commonly used F-techniques in developmental biology: Fluorescence Recovery After Photo-bleaching (FRAP), Fo¨ rster Resonance Energy Transfer (FRET), and Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS and FCCS).

Developmentally inspired human 'organs on chips'.

Although initially developed to replace animal testing in drug development, human 'organ on a chip' (organ chip) microfluidic culture technology offers a new tool for studying tissue development and pathophysiology, which has brought us one step closer to carrying out human experimentation in vitro In this Spotlight article, I discuss the central role that developmental biology played in the early stages of organ-chip technology, and how these models have led to new insights into human physiology and disease mechanisms. Advantages and disadvantages of the organ-chip approach relative to organoids and other human cell cultures are also discussed.

Illuminating developmental biology through photochemistry.

Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanisms. Most recently, genome sequencing and 'omics' profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this Perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through the use of these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.

Taking the strain: quantifying the contributions of all cell behaviours to changes in epithelial shape.

Computer-assisted tracking of the shapes of many cells over long periods of development has driven the exploration of novel ways to quantify the contributions of different cell behaviours to morphogenesis. A handful of similar methods have now been published that are used to calculate tissue deformations (strain rates) in epithelia. These methods are further used to quantify strain rates attributable to each of the cell behaviours in the tissue, such as cell shape change, cell rearrangement and cell division, that together sum to the tissue strain rates. In this review, aimed at developmental biologists, I will introduce the general approach, characterize differences in current approaches and highlight extensions of these methods that remain to be fully explored. The methods will make a major contribution to the emerging field of tissue mechanics. Precisely quantified strain rates are an essential first step towards exploring constitutive equations relating stress to strain via tissue mechanical properties.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.

Deciphering tissue morphodynamics using bioimage informatics.

In recent years developmental biology has greatly benefited from the latest advances in fluorescence microscopy techniques. Consequently, quantitative and automated analysis of this data is becoming a vital first step in the quest for novel insights into the various aspects of development. Here we present an introductory overview of the various image analysis methods proposed for developmental biology images, with particular attention to openly available software packages. These tools, as well as others to come, are rapidly paving the way towards standardized and reproducible bioimaging studies at the whole-tissue level. Reflecting on these achievements, we discuss the remaining challenges and the future endeavours lying ahead in the post-image analysis era.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.

Inferring cellular forces from image stacks.

Although the importance of cellular forces to a wide range of embryogenesis and disease processes is widely recognized, measuring these forces is challenging, especially in three dimensions. Here, we introduce CellFIT-3D, a force inference technique that allows tension maps for three-dimensional cellular systems to be estimated from image stacks. Like its predecessors, video force microscopy and CellFIT, this cell mechanics technique assumes boundary-specific interfacial tensions to be the primary drivers, and it constructs force-balance equations based on triple junction (TJ) dihedral angles. The technique involves image processing, segmenting of cells, grouping of cell outlines, calculation of dihedral planes, averaging along three-dimensional TJs, and matrix equation assembly and solution. The equations tend to be strongly overdetermined, allowing indistinct TJs to be ignored and solution error estimates to be determined. Application to clean and noisy synthetic data generated using Surface Evolver gave tension errors of 1.6-7%, and analyses of eight-cell murine embryos gave estimated errors smaller than the 10% uncertainty of companion aspiration experiments. Other possible areas of application include morphogenesis, cancer metastasis and tissue engineering.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.

zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.

Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.

Segmentation and quantitative analysis of individual cells in developmental tissues.

Image analysis is vital for extracting quantitative information from biological images and is used extensively, including investigations in developmental biology. The technique commences with the segmentation (delineation) of objects of interest from 2D images or 3D image stacks and is usually followed by the measurement and classification of the segmented objects. This chapter focuses on the segmentation task and here we explain the use of ImageJ, MIPAV (Medical Image Processing, Analysis, and Visualization), and VisSeg, three freely available software packages for this purpose. ImageJ and MIPAV are extremely versatile and can be used in diverse applications. VisSeg is a specialized tool for performing highly accurate and reliable 2D and 3D segmentation of objects such as cells and cell nuclei in images and stacks.

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging.

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, "3D HiLo" where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Light sheet microscopy for real-time developmental biology.

Within only a few short years, light sheet microscopy has contributed substantially to the emerging field of real-time developmental biology. Low photo-toxicity and high-speed multiview acquisition have made selective plane illumination microscopy (SPIM) a popular choice for studies of organ morphogenesis and function in zebrafish, Drosophila, and other model organisms. A multitude of different light sheet microscopes have emerged for the noninvasive imaging of specimens ranging from single molecules to cells, tissues, and entire embryos. In particular, developmental biology can benefit from the ability to watch developmental events occur in real time in an entire embryo, thereby advancing our understanding on how cells form tissues and organs. However, it presents a new challenge to our existing data and image processing tools. This review gives an overview of where we stand as light sheet microscopy branches out, explores new areas, and becomes more specialized.

Bridging structure and process in developmental biology through new imaging technologies.

Many unexpected discoveries in developmental biology have depended on advancement of imaging technologies to visualize developmental processes as they unfold across multiple spatial and temporal scales. This essay surveys the recent advances in imaging, highlighting emerging capabilities with an eye toward those poised to have the greatest impact on developmental biology.

Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults.

Reconstructing embryonic development.

Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources.

Lighting up developmental mechanisms: how fluorescence imaging heralded a new era.

Embryology and genetics have given rise to a mechanistic framework that explains the architecture of a developing organism. Until recently, however, such studies suffered from a lack of quantification and real-time visualization at the subcellular level, limiting their ability to monitor the dynamics of developmental processes. Live imaging using fluorescent proteins has overcome these limitations, uncovering unprecedented insights that call many established models into question. We review how the study of patterning, cell polarization and morphogenesis has benefited from this technology and discuss the possibilities offered by fluorescence imaging and by the contributions of quantitative disciplines.

Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses.

Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

A standard system to study vertebrate embryos.

Staged embryonic series are important as reference for different kinds of biological studies. I summarise problems that occur when using 'staging tables' of 'model organisms'. Investigations of developmental processes in a broad scope of taxa are becoming commonplace. Beginning in the 1990s, methods were developed to quantify and analyse developmental events in a phylogenetic framework. The algorithms associated with these methods are still under development, mainly due to difficulties of using non-independent characters. Nevertheless, the principle of comparing clearly defined newly occurring morphological features in development (events) in quantifying analyses was a key innovation for comparative embryonic research. Up to date no standard was set for how to define such events in a comparative approach. As a case study I compared the external development of 23 land vertebrate species with a focus on turtles, mainly based on reference staging tables. I excluded all the characters that are only identical for a particular species or general features that were only analysed in a few species. Based on these comparisons I defined 104 developmental characters that are common either for all vertebrates (61 characters), gnathostomes (26), tetrapods (3), amniotes (7), or only for sauropsids (7). Characters concern the neural tube, somite, ear, eye, limb, maxillary and mandibular process, pharyngeal arch, eyelid or carapace development. I present an illustrated guide listing all the defined events. This guide can be used for describing developmental series of any vertebrate species or for documenting specimen variability of a particular species. The guide incorporates drawings and photographs as well as consideration of species identifying developmental features such as colouration. The simple character-code of the guide is extendable to further characters pertaining to external and internal morphological, physiological, genetic or molecular development, and also for other vertebrate groups not examined here, such as Chondrichthyes or Actinopterygii. An online database to type in developmental events for different stages and species could be a basis for further studies in comparative embryology. By documenting developmental events with the standard code, sequence heterochrony studies (i.e. Parsimov) and studies on variability can use this broad comparative data set.