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      High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

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          Abstract

          The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology.

          Abstract

          Systematic large-scale analysis of embryonic development requires the processing of large amounts of microscopy data. Here Schmid et al. solve this problem by developing a high-speed imaging system that projects zebrafish embryos onto a ‘world map’ in real time, revealing characteristic migration patterns in the early endoderm.

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          Most cited references15

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          Selective plane illumination microscopy techniques in developmental biology.

          Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections. In addition, high depth penetration, low bleaching and high acquisition speeds make light-sheet microscopy ideally suited for extended time-lapse experiments in live embryos. This review compares the benefits and challenges of light-sheet microscopy with established fluorescence microscopy techniques such as confocal microscopy and discusses the different implementations and applications of this easily adaptable technology.
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            A high-level 3D visualization API for Java and ImageJ

            Background Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set. Results Here we present a platform-independent framework based on Java and Java 3D for accelerated rendering of biological images. Our framework is seamlessly integrated into ImageJ, a free image processing package with a vast collection of community-developed biological image analysis tools. Our framework enriches the ImageJ software libraries with methods that greatly reduce the complexity of developing image analysis tools in an interactive 3D visualization environment. In particular, we provide high-level access to volume rendering, volume editing, surface extraction, and image annotation. The ability to rely on a library that removes the low-level details enables concentrating software development efforts on the algorithm implementation parts. Conclusions Our framework enables biomedical image software development to be built with 3D visualization capabilities with very little effort. We offer the source code and convenient binary packages along with extensive documentation at http://3dviewer.neurofly.de.
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              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).
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Pub. Group
                2041-1723
                25 July 2013
                : 4
                : 2207
                Affiliations
                [1 ]Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108 , 01307 Dresden, Germany
                [2 ]Institute for Medical Informatics and Biometry, Medical School, TU Dresden, Fetscherstr. 74 , 01307 Dresden, Germany
                [3 ]Institute of Molecular Biology and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Technikerstr. 25 , A-6020 Innsbruck, Austria
                Author notes
                Article
                ncomms3207
                10.1038/ncomms3207
                3731668
                23884240
                96c0a44f-7c9d-4a2d-a7ac-a66f1a74b7e4
                Copyright © 2013, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                History
                : 08 January 2013
                : 28 June 2013
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