Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy

Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS, INSERM, Palaiseau, France.
Science (Impact Factor: 33.61). 08/2010; 329(5994):967-71. DOI: 10.1126/science.1189428
Source: PubMed


Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated
image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10
cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal
accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided
intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology
of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens
according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule,
contrasting with classical descriptions of abrupt changes in the system dynamics.

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    • "The lack of robust, fast and universal procedures to provide such a geometric description is probably one of the main obstacles to exploit the full potential of images. The mainstream approach to analyze image contents nowadays consists in segmenting each cell/nuclei independently [22] [24]. A precise segmentation completely describes the geometrical contents of images and is often regarded as the best source of information one can hope for. "
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    ABSTRACT: Motivation: Extracting geometrical information from large 2D or 3D biomedical images is important to better understand fundamental phenomena such as morphogenesis. We address the problem of automatically analyzing spatial organization of cells or nuclei in 2D or 3D images of tissues. This problem is challenging due to the usually low quality of microscopy images as well as their typically large sizes. Results: The structure tensor is a simple and robust descriptor that was developed to analyze textures orientation. Contrarily to segmentation methods which rely on an object based modelling of images, the structure tensor views the sample at a macroscopic scale, like a continuum. We propose an original theoretical analysis of this tool and show that it allows quantifying two important features of nuclei in tissues: their privileged orientation as well as the ratio between the length of their main axes. A quantitative evaluation of the method is provided for synthetic and real 2D and 3D images. As an application, we analyze the nuclei orientation and anisotropy on multicellular tumor spheroids cryosections. This analysis reveals that cells are elongated in a privileged direction that is parallel to the boundary of the spheroid. Availability: Source codes are available at
    07/2014; DOI:10.1109/TMI.2015.2470093
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    • "Using this approach, quantitative measurements of 3D cell shape have been demonstrated in the plant meristem (Fernandez et al., 2010; Federici et al., 2012). In metazoans, tools have focused on early developmental stages of zebrafish and ascidians, the morphologies of which are relatively simple and in which cell movement is limited (Olivier et al., 2010; Sherrard et al., 2010). For processes that display greater morphological changes, cell shape is often ignored and nuclei are used as a proxy for cell position (McMahon et al., 2008; Giurumescu et al., 2012). "
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    ABSTRACT: Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.
    Development 06/2014; 141(14). DOI:10.1242/dev.107730 · 6.46 Impact Factor
    • "Second and third harmonic generation (SHG and THG) are non-destructive imaging techniques that have been extensively used as diagnostic tools for biological studies (Sun et al 2004, Debarre et al 2006, Luo et al 2009, Zhuo et al 2009, Olivier et al 2010, Tserevelakis et al 2011, Xiong et al 2011). Recently, THG microscopy has been employed for monitoring laser–polymer interactions (Selimis et al 2012), providing very promising results. "
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    ABSTRACT: Insight into the processes taking place upon ultraviolet (UV) laser ablation of polymers is of high importance. In this study, a new algorithm for the precise analysis of third harmonic generation (THG) images from irradiated polymers has been developed. In particular, high accuracy qualitative and quantitative morphological information concerning the induced swelling following UV laser irradiation has been obtained by employing the proposed algorithm. Furthermore, compensation for the curvature of the field, correction of the apparent depth distortion and delineation capability of the total swelling area at a high resolution has been achieved. The above advantages allow a precise and comprehensive monitoring (via the detection of THG signals) of the dynamics during the interaction of laser radiation with polymeric materials to an extent that is not possible with other standard techniques such as profilometry or electron microscopy.
    Laser Physics 11/2013; 23(12):126005-7. DOI:10.1088/1054-660X/23/12/126005 · 1.03 Impact Factor
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