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

ABSTRACT 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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    • "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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    • "The biology of cells and tissues is now investigated in detail in terms of protein distribution and gene expression, especially during development [11]. It is possible to image the full geometry of an embryonic tissue at cellular resolution [12] [13], while simultaneously visualising the expression of various genes of interest [14] [15] [16]. Mechanical fields such as the deformation, deformation rate or plastic deformation rate are increasingly accessible to direct measurement. "
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    ABSTRACT: The understanding of morphogenesis in living organisms has been renewed by tremendous progress in experimental techniques that provide access to cell-scale, quantitative information both on the shapes of cells within tissues and on the genes being expressed. This information suggests that our understanding of the respective contributions of gene expression and mechanics, and of their crucial entanglement, will soon leap forward. Biomechanics increasingly benefits from models, which assist the design and interpretation of experiments, point out the main ingredients and assumptions, and can ultimately lead to predictions. The newly accessible local information thus urges for a reflection on how to select suitable classes of mechanical models. We review both mechanical ingredients suggested by the current knowledge of tissue behaviour, and modelling methods that can help generate a constitutive equation. We also recall the mathematical framework developped for continuum materials and how to transform a constitutive equation into a system of partial differential equations amenable to numerical resolution. The present article thus groups together mechanical elements and theoretical methods that are ready to enhance the significance of the data extracted from recent or future high throughput biomechanical experiments.
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    • "THG is created also in inversion symmetric structures, but due to the significantly different refractive index at the THG wavelength, the coherent superposition oscillates in space and is suppressed away from interfaces or structures having sizes comparable to the excitation wavelength [7] [8]. Since lipids have a much higher nonlinear susceptibility [9] [10] for THG than water, this microscopy modality is useful for imaging lipid-rich structures [11] [12]. On the other hand, SHG and THG do not provide vibrational contrast, since the involved light frequencies are well above the vibrational frequency range. "
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    ABSTRACT: Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.
    Progress in lipid research 09/2013; 52(4). DOI:10.1016/j.plipres.2013.07.003 · 10.02 Impact Factor
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