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3 Janelia Publications

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    06/24/12 | Tandem fluorescent protein timers for in vivo analysis of protein dynamics.
    Khmelinskii A, Keller PJ, Bartosik A, Meurer M, Barry JD, Mardin BR, Kaufmann A, Trautmann S, Wachsmuth M, Pereira G, Huber W, Schiebel E, Knop M
    Nature Biotechnology. 2012 Jun 24;30(7):708-14. doi: 10.1038/nbt.2281

    The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

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    06/03/12 | Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy.
    Tomer R, Khairy K, Amat F, Keller PJ
    Nature Methods. 2012 Jun 3;9(7):755-63. doi: 10.1038/nmeth.2062

    Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

    High-resolution movies in the Digital Embryo repository
    Nature News: "Fruitfly development, cell by cell" by Lauren Gravitz
    Nature Methods Technology Feature: "Faster frames, clearer pictures" by Monya Baker
    Andor Insight Awards: Life Sciences Winner

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    02/01/12 | Light sheet microscopy of living or cleared specimens.
    Keller PJ, Dodt H
    Current Opinion in Neurobiology. 2012 Feb;22(1):138-43. doi: 10.1016/j.conb.2011.08.003

    Light sheet microscopy is a versatile imaging technique with a unique combination of capabilities. It provides high imaging speed, high signal-to-noise ratio and low levels of photobleaching and phototoxic effects. These properties are crucial in a wide range of applications in the life sciences, from live imaging of fast dynamic processes in single cells to long-term observation of developmental dynamics in entire large organisms. When combined with tissue clearing methods, light sheet microscopy furthermore allows rapid imaging of large specimens with excellent coverage and high spatial resolution. Even samples up to the size of entire mammalian brains can be efficiently recorded and quantitatively analyzed. Here, we provide an overview of the history of light sheet microscopy, review the development of tissue clearing methods, and discuss recent technical breakthroughs that have the potential to influence the future direction of the field.

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