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60 Janelia Publications

Showing 31-40 of 60 results
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    Looger LabKeller Lab
    12/15/15 | Direct in vivo manipulation and imaging of calcium transients in neutrophils identify a critical role for leading-edge calcium flux.
    Beerman RW, Matty MA, Au GG, Looger LL, Choudhury KR, Keller PJ, Tobin DM
    Cell Reports. 2015 Dec 15;13(10):2107-17. doi: 10.1016/j.celrep.2015.11.010

    Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

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    11/02/15 | Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.
    Kim S, Cho JH, Murray E, Bakh N, Choi H, Ohn K, Ruelas L, Hubbert A, McCue M, Vassallo SL, Keller PJ, Chung K
    Proceedings of the National Academy of Sciences of the United States of America. 2015 Nov 2;112(46):E6274-83. doi: 10.1073/pnas.1510133112

    Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1–3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.

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    10/26/15 | Whole-animal functional and developmental imaging with isotropic spatial resolution
    Chhetri RK, Amat F, Wan Y, Höckendorf B, Lemon WC, Keller PJ
    Nature Methods. 2015 Oct 26;12(12):1171-8. doi: 10.1038/nmeth.3632

    Imaging fast cellular dynamics across large specimens requires high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To meet these requirements, we developed isotropic multiview (IsoView) light-sheet microscopy, which rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. Combining these four views by means of high-throughput multiview deconvolution yields images with high resolution in all three dimensions. We demonstrate whole-animal functional imaging of Drosophila larvae at a spatial resolution of 1.1-2.5 μm and temporal resolution of 2 Hz for several hours. We also present spatially isotropic whole-brain functional imaging in Danio rerio larvae and spatially isotropic multicolor imaging of fast cellular dynamics across gastrulating Drosophila embryos. Compared with conventional light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

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    10/02/15 | Efficient processing and analysis of large-scale light-sheet microscopy data.
    Amat F, Höckendorf B, Wan Y, Lemon WC, McDole K, Keller PJ
    Nature Protocols. 2015 Oct 2;10(11):1679-96. doi: 10.1038/nprot.2015.111

    Light-sheet microscopy is a powerful method for imaging the development and function of complex biological systems at high spatiotemporal resolution and over long time scales. Such experiments typically generate terabytes of multidimensional image data, and thus they demand efficient computational solutions for data management, processing and analysis. We present protocols and software to tackle these steps, focusing on the imaging-based study of animal development. Our protocols facilitate (i) high-speed lossless data compression and content-based multiview image fusion optimized for multicore CPU architectures, reducing image data size 30–500-fold; (ii) automated large-scale cell tracking and segmentation; and (iii) visualization, editing and annotation of multiterabyte image data and cell-lineage reconstructions with tens of millions of data points. These software modules are open source. They provide high data throughput using a single computer workstation and are readily applicable to a wide spectrum of biological model systems.

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    08/11/15 | Whole-central nervous system functional imaging in larval Drosophila.
    Lemon WC, Pulver SR, Höckendorf B, McDole K, Branson KM, Freeman J, Keller PJ
    Nature Communications. 2015 Aug 11;6:7924. doi: 10.1038/ncomms8924

    Understanding how the brain works in tight concert with the rest of the central nervous system (CNS) hinges upon knowledge of coordinated activity patterns across the whole CNS. We present a method for measuring activity in an entire, non-transparent CNS with high spatiotemporal resolution. We combine a light-sheet microscope capable of simultaneous multi-view imaging at volumetric speeds 25-fold faster than the state-of-the-art, a whole-CNS imaging assay for the isolated Drosophila larval CNS and a computational framework for analysing multi-view, whole-CNS calcium imaging data. We image both brain and ventral nerve cord, covering the entire CNS at 2 or 5 Hz with two- or one-photon excitation, respectively. By mapping network activity during fictive behaviours and quantitatively comparing high-resolution whole-CNS activity maps across individuals, we predict functional connections between CNS regions and reveal neurons in the brain that identify type and temporal state of motor programs executed in the ventral nerve cord.

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    04/16/15 | Interactive exemplar-based segmentation toolkit for biomedical image analysis.
    Li X, Zhou Z, Keller PJ, Zeng H, Liu T, Peng H
    International Symposium on Biomedical Imaging. 2015 Apr:

    In the field of biomedical imaging analysis on single-cell level, reliable and fast segmentation of the cell nuclei from the background on three-dimensional images is highly needed for the further analysis. In this work we propose an interactive cell segmentation toolkit that first establishes a set of exemplar regions from user input through an easy and intuitive interface in both 2D and 3D in real-time, then
    extracts the shape and intensity features from those exemplars. Based on a local contrast-constrained region growing scheme, each connected component in the whole image would be filtered by the features from exemplars, forming an “exemplar-matching” group which passed the filtering and would be part of the final segmentation result, and a “non-exemplar-matching” group in which components
    would be further segmented by the gradient vector field based algorithm. The results of the filtering process are visualized back to the user in near real-time, thus enhancing the experience in exemplar selecting and parameter tuning. The toolkit is distributed as a plugin within the open source Vaa3D system (http://vaa3d.org).

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    02/04/15 | Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy.
    Keller PJ, Ahrens MB
    Neuron. 2015 Feb 4;85(3):462-83. doi: 10.1016/j.neuron.2014.12.039

    The nature of nervous system function and development is inherently global, since all components eventually influence one another. Networks communicate through dense synaptic, electric, and modulatory connections and develop through concurrent growth and interlinking of their neurons, processes, glia, and blood vessels. These factors drive the development of techniques capable of imaging neural signaling, anatomy, and developmental processes at ever-larger scales. Here, we discuss the nature of questions benefitting from large-scale imaging techniques and introduce recent applications. We focus on emerging light-sheet microscopy approaches, which are well suited for live imaging of large systems with high spatiotemporal resolution and over long periods of time. We also discuss computational methods suitable for extracting biological information from the resulting system-level image data sets. Together with new tools for reporting and manipulating neuronal activity and gene expression, these techniques promise new insights into the large-scale function and development of neural systems.

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    12/30/14 | Light-sheet imaging for systems neuroscience.
    Keller PJ, Ahrens MB, Freeman J
    Nature Methods. 2014 Dec 30;12(1):27-9. doi: 10.1038/nmeth.3214

    Developments in electrical and optical recording technology are scaling up the size of neuronal populations that can be monitored simultaneously. Light-sheet imaging is rapidly gaining traction as a method for optically interrogating activity in large networks and presents both opportunities and challenges for understanding circuit function.

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    11/07/14 | Making biology transparent.
    Höckendorf B, Lavis LD, Keller PJ
    Nature Biotechnology. 2014 Nov 7;32(11):1104-5. doi: 10.1038/nbt.3061

    The molecular and cellular architecture of the organs in a whole mouse is revealed through optical clearing.

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    10/15/14 | The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.
    Crespo C, Vernieri C, Keller PJ, Garrè M, Bender JR, Wittbrodt J, Pardi R
    Journal of Cell Science. 2014 Oct 15;127(Pt 20):4381-95. doi: 10.1242/jcs.146217

    Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

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