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60 Janelia Publications

Showing 41-50 of 60 results
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    09/12/14 | Development of the annelid axochord: insights into notochord evolution.
    Lauri A, Brunet T, Handberg-Thorsager M, Fischer AH, Simakov O, Steinmetz PR, Tomer R, Keller PJ, Arendt D
    Science. 2014 Sep 12;345(6202):1365-8. doi: 10.1126/science.1253396

    The origin of chordates has been debated for more than a century, with one key issue being the emergence of the notochord. In vertebrates, the notochord develops by convergence and extension of the chordamesoderm, a population of midline cells of unique molecular identity. We identify a population of mesodermal cells in a developing invertebrate, the marine annelid Platynereis dumerilii, that converges and extends toward the midline and expresses a notochord-specific combination of genes. These cells differentiate into a longitudinal muscle, the axochord, that is positioned between central nervous system and axial blood vessel and secretes a strong collagenous extracellular matrix. Ancestral state reconstruction suggests that contractile mesodermal midline cells existed in bilaterian ancestors. We propose that these cells, via vacuolization and stiffening, gave rise to the chordate notochord.

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    08/22/14 | Light sheet-based imaging and analysis of early embryogenesis in the fruit fly.
    Khairy K, Lemon WC, Amat F, Keller PJ
    Methods in Molecular Biology. 2015;1189:79-97. doi: 10.1007/978-1-4939-1164-6_6

    The fruit fly is an excellent model system for investigating the sequence of epithelial tissue invaginations constituting the process of gastrulation. By combining recent advancements in light sheet fluorescence microscopy (LSFM) and image processing, the three-dimensional fly embryo morphology and relevant gene expression patterns can be accurately recorded throughout the entire process of embryogenesis. LSFM provides exceptionally high imaging speed, high signal-to-noise ratio, low level of photoinduced damage, and good optical penetration depth. This powerful combination of capabilities makes LSFM particularly suitable for live imaging of the fly embryo.The resulting high-information-content image data are subsequently processed to obtain the outlines of cells and cell nuclei, as well as the geometry of the whole embryo tissue by image segmentation. Furthermore, morphodynamics information is extracted by computationally tracking objects in the image. Towards that goal we describe the successful implementation of a fast fitting strategy of Gaussian mixture models.The data obtained by image processing is well-suited for hypothesis testing of the detailed biomechanics of the gastrulating embryo. Typically this involves constructing computational mechanics models that consist of an objective function providing an estimate of strain energy for a given morphological configuration of the tissue, and a numerical minimization mechanism of this energy, achieved by varying morphological parameters.In this chapter, we provide an overview of in vivo imaging of fruit fly embryos using LSFM, computational tools suitable for processing the resulting images, and examples of computational biomechanical simulations of fly embryo gastrulation.

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    Ahrens LabLooger LabKeller LabFreeman Lab
    07/27/14 | Light-sheet functional imaging in fictively behaving zebrafish.
    Vladimirov N, Mu Y, Kawashima T, Bennett DV, Yang C, Looger LL, Keller PJ, Freeman J, Ahrens MB
    Nature Methods. 2014 Jul 27;11(9):883-4. doi: 10.1038/nmeth.3040

    The processing of sensory input and the generation of behavior involves large networks of neurons, which necessitates new technology for recording from many neurons in behaving animals. In the larval zebrafish, light-sheet microscopy can be used to record the activity of almost all neurons in the brain simultaneously at single-cell resolution. Existing implementations, however, cannot be combined with visually driven behavior because the light sheet scans over the eye, interfering with presentation of controlled visual stimuli. Here we describe a system that overcomes the confounding eye stimulation through the use of two light sheets and combines whole-brain light-sheet imaging with virtual reality for fictively behaving larval zebrafish.

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    07/20/14 | Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data.
    Amat F, Lemon W, Mossing DP, McDole K, Wan Y, Branson K, Myers EW, Keller PJ
    Nature Methods. 2014 Jul 20;11(9):951-8. doi: 10.1038/nmeth.3036

    The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min(-1) on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

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    09/16/13 | Live imaging of nervous system development and function using light-sheet microscopy.
    Lemon WC, Keller PJ
    Molecular Reproduction and Development. 2015 Jul;82(7-8):605-18. doi: 10.1002/mrd.22258

    In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

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    08/15/13 | In vivo imaging of zebrafish embryogenesis.
    Keller PJ
    Methods. 2013 Aug 15;62(3):268-78. doi: 10.1016/j.ymeth.2013.03.015

    The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis. Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key properties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms.

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    06/07/13 | Imaging morphogenesis: technological advances and biological insights.
    Keller PJ
    Science. 2013 Jun 7;340(6137):1234168. doi: 10.1126/science.1234168

    Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

    Science Profile: A Research Career in Focus

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    05/01/13 | Whole-brain functional imaging at cellular resolution using light-sheet microscopy.
    Ahrens MB, Orger MB, Robson DN, Li JM, Keller PJ
    Nature Methods. 2013 May;10(5):413-20. doi: 10.1038/nmeth.2434

    Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

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    04/01/13 | 3D Haar-like elliptical features for object classification in microscopy.
    Amat F, Keller PJ
    International Symposium on Biomedical Imaging. 2013 Apr:

    Object detection and classification are key tasks in computer vision that can facilitate high-throughput image analysis of microscopy data. We present a set of local image descriptors for three-dimensional (3D) microscopy datasets inspired by the well-known Haar wavelet framework. We add orientation, illumination and scale information by assuming that the neighborhood surrounding points of interests in the image can be described with ellipsoids, and we increase discriminative power by incorporating edge and shape information into the features. The calculation of the local image descriptors is implemented in a Graphics Processing Unit (GPU) in order to reduce computation time to 1 millisecond per object of interest. We present results for cell division detection in 3D time-lapse fluorescence microscopy with 97.6% accuracy.

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    04/29/13 | Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.
    Amat F, Keller PJ
    Development, Growth and Differentiation. 2013 Apr 29;55(4):563-78. doi: 10.1111/dgd.12063

    Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse.

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