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2432 Janelia Publications

Showing 2031-2040 of 2432 results
01/01/13 | Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.
Abrahamsson S, Chen J, Hajj B, Stallinga S, Katsov AY, Wisniewski J, Mizuguchi G, Soule P, Mueller F, Darzacq CD, Darzacq X, Wu C, Bargmann CI, Agard DA, Dahan M, Gustafsson MG
Nature Methods. 2013;10(1):60-3. doi: 10.1038/nmeth.2277

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

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Cui Lab
01/01/13 | High speed phase distortion measurement and compensation for focusing in space and time.
Fiolka R, Cui M
Proceedings of SPIE. 2013;8589:85890V. doi: 10.1117/12.2001121

Random scattering and aberrations severely limit the imaging depth in optical microscopy. We introduce a rapid, parallel wavefront compensation technique that efficiently compensates even highly complex phase distortions. Using coherence gated backscattered light as a feedback signal, we focus light deep inside highly scattering brain tissue. We demonstrate that the same wavefront optimization technique can also be used to compensate spectral phase distortions in ultrashort laser pulses using nonlinear iterative feedback. We can restore transform limited pulse durations at any selected target location and compensate for dispersion that has occurred in the optical train and within the sample.

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01/01/13 | Light sheet microscopy in cell biology.
Tomer R, Khairy K, Keller PJ
Methods in Molecular Biology. 2013;931:123-37. doi: 10.1007/978-1-62703-056-4_7

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching, and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. Here, we provide an overview of light sheet-based microscopy assays for in vitro and in vivo imaging of biological samples, including cell extracts, soft gels, and large multicellular organisms. We furthermore describe computational tools for basic image processing and data inspection.

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Riddiford Lab
10/09/13 | Nancy E. Beckage 1950-2012: Pioneer in insect host-parasite interactions.
Riddiford LM, Webb BA
Annual Review of Entomology. 2013 Oct 9;59:1-12. doi: 10.1146/annurev-ento-052913-021246

Nancy E. Beckage is widely recognized for her pioneering work in the field of insect host-parasitoid interactions beginning with endocrine influences of the tobacco hornworm, Manduca sexta, host and its parasitoid wasp Apanteles congregatus (now Cotesia congregata) on each other’s development. Moreover, her studies show that the polydnavirus carried by the parasitoid wasp not only protects the parasitoid from the host’s immune defenses, but also is responsible for some of the developmental effects of parasitism. Nancy was a highly regarded mentor of both undergraduate and graduate students and more widely of women students and colleagues in entomology. Her service both to her particular area and to entomology in general through participation on federal grant review panels and in the governance of the Entomological Society of America, organization of symposia at both national and international meetings, and editorship of several different journal issues and of several books, is legendary. She has left behind a lasting legacy of increased understanding of multilevel endocrine and physiological interactions among insects and other organisms and a strong network of interacting scientists and colleagues in her area of entomology.

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01/01/13 | Neural signals of extinction in the inhibitory microcircuit of the ventral midbrain.
Pan W, Brown J, Dudman JT
Nature Neuroscience. 2013 Jan;16(1):71-8. doi: 10.1038/nn.3283

Midbrain dopaminergic (DA) neurons are thought to guide learning via phasic elevations of firing in response to reward predicting stimuli. The mechanism for these signals remains unclear. Using extracellular recording during associative learning, we found that inhibitory neurons in the ventral midbrain of mice responded to salient auditory stimuli with a burst of activity that occurred before the onset of the phasic response of DA neurons. This population of inhibitory neurons exhibited enhanced responses during extinction and was anticorrelated with the phasic response of simultaneously recorded DA neurons. Optogenetic stimulation revealed that this population was, in part, derived from inhibitory projection neurons of the substantia nigra that provide a robust monosynaptic inhibition of DA neurons. Thus, our results elaborate on the dynamic upstream circuits that shape the phasic activity of DA neurons and suggest that the inhibitory microcircuit of the midbrain is critical for new learning in extinction.

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01/01/13 | Rfam 11.0: 10 years of RNA families.
Burge SW, Daub J, Eberhardt R, Tate J, Barquist L, Nawrocki EP, Eddy SR, Gardner PP, Bateman A
Nucleic Acids Research. 2013 Jan;41:D226-32. doi: 10.1093/nar/gks1005

The Rfam database (available via the website at http://rfam.sanger.ac.uk and through our mirror at http://rfam.janelia.org) is a collection of non-coding RNA families, primarily RNAs with a conserved RNA secondary structure, including both RNA genes and mRNA cis-regulatory elements. Each family is represented by a multiple sequence alignment, predicted secondary structure and covariance model. Here we discuss updates to the database in the latest release, Rfam 11.0, including the introduction of genome-based alignments for large families, the introduction of the Rfam Biomart as well as other user interface improvements. Rfam is available under the Creative Commons Zero license.

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01/01/13 | The chemistry of small-molecule fluorogenic probes.
Grimm JB, Heckman LM, Lavis LD
Progress in Molecular Biology and Translational Science;113:1-34. doi: 10.1016/B978-0-12-386932-6.00001-6

Chemical fluorophores find wide use in biology to detect and visualize different phenomena. A key advantage of small-molecule dyes is the ability to construct compounds where fluorescence is activated by chemical or biochemical processes. Fluorogenic molecules, in which fluorescence is activated by enzymatic activity, light, or environmental changes, enable advanced bioassays and sophisticated imaging experiments. Here, we detail the collection of fluorophores and highlight both general strategies and unique approaches that are employed to control fluorescence using chemistry.

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Gonen Lab
01/01/13 | The collection of high-resolution electron diffraction data.
Gonen T
Methods in Molecular Biology. 2013;955:153-169. doi: 10.1007/978-1-62703-176-9_9

A number of atomic-resolution structures of membrane proteins (better than 3Å resolution) have been determined recently by electron crystallography. While this technique was established more than 40 years ago, it is still in its infancy with regard to the two-dimensional (2D) crystallization, data collection, data analysis, and protein structure determination. In terms of data collection, electron crystallography encompasses both image acquisition and electron diffraction data collection. Other chapters in this volume outline protocols for image collection and analysis. This chapter, however, outlines detailed protocols for data collection by electron diffraction. These include microscope setup, electron diffraction data collection, and troubleshooting.

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Bock Lab
06/18/13 | The Open Connectome Project Data Cluster: Scalable analysis and vision for high-throughput neuroscience.
Burns R, Roncal WG, Kleissas D, Lillaney K, Manavalan P, Perlman E, Berger DR, Bock DD, Chung K, Grosenick L, Kasthuri N, Weiler NC, Deisseroth K, Kazhdan M, Lichtman J, Reid RC, Smith SJ, Szalay AS, Vogelstein JT, Vogelstein RJ
Scientific and Statistical Database Management: International Conference, SSDBM ... : Proceedings. International Conference on Scientific and Statistical Database Management. 2013 Jun 18:. doi: 10.1145/2484838.2484870

We describe a scalable database cluster for the spatial analysis and annotation of high-throughput brain imaging data, initially for 3-d electron microscopy image stacks, but for time-series and multi-channel data as well. The system was designed primarily for workloads that build connectomes- neural connectivity maps of the brain-using the parallel execution of computer vision algorithms on high-performance compute clusters. These services and open-science data sets are publicly available at openconnecto.me. The system design inherits much from NoSQL scale-out and data-intensive computing architectures. We distribute data to cluster nodes by partitioning a spatial index. We direct I/O to different systems-reads to parallel disk arrays and writes to solid-state storage-to avoid I/O interference and maximize throughput. All programming interfaces are RESTful Web services, which are simple and stateless, improving scalability and usability. We include a performance evaluation of the production system, highlighting the effec-tiveness of spatial data organization.

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01/01/13 | The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.
Swart EC, Bracht JR, Magrini V, Minx P, Chen X, Zhou Y, Khurana JS, Goldman AD, Nowacki M, Schotanus K, Jung S, Fulton RS, Ly A, McGrath S, Haub K, Wiggins JL, Storton D, Matese JC, Parsons L, Chang W, Bowen MS, Stover NA, Jones TA, Eddy SR, Herrick GA, Doak TG, Wilson RK, Mardis ER, Landweber LF
PLoS Biology. 2013 Jan;11(1):e1001473. doi: 10.1371/journal.pbio.1001473

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (\~{}5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of \~{}2,000 copies. We report the high-quality genome assembly of \~{}16,000 complete nanochromosomes (\~{}50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean \~{}3.2 kb) and encode \~{}18,500 genes. Alternative DNA fragmentation processes \~{}10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is \~{}4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.

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