Nature Methods

Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.

Publication Date: 2012 May 13 PMID: 22581372
Authors: York, A. G. - Parekh, S. H. - Nogare, D. D. - Fischer, R. S. - Temprine, K. - Mione, M. - Chitnis, A. B. - Combs, C. A. - Shroff, H.
Journal: Nat Methods

We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 mum. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

post to: CiteULike

M-Track: detecting short-lived protein-protein interactions in vivo.

Publication Date: 2012 May 13 PMID: 22581371
Authors: Zuzuarregui, A. - Kupka, T. - Bhatt, B. - Dohnal, I. - Mudrak, I. - Friedmann, C. - Schuchner, S. - Frohner, I. E. - Ammerer, G. - Ogris, E.
Journal: Nat Methods

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

post to: CiteULike

Rational design of true monomeric and bright photoactivatable fluorescent proteins.

Publication Date: 2012 May 13 PMID: 22581370
Authors: Zhang, M. - Chang, H. - Zhang, Y. - Yu, J. - Wu, L. - Ji, W. - Chen, J. - Liu, B. - Lu, J. - Liu, Y. - Zhang, J. - Xu, P. - Xu, T.
Journal: Nat Methods

Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

post to: CiteULike

Simultaneous BOLD fMRI and fiber-optic calcium recording in rat neocortex.

Publication Date: 2012 May 6 PMID: 22561989
Authors: Schulz, K. - Sydekum, E. - Krueppel, R. - Engelbrecht, C. J. - Schlegel, F. - Schroter, A. - Rudin, M. - Helmchen, F.
Journal: Nat Methods

Functional magnetic resonance imaging (fMRI) based on blood oxygen level-dependent (BOLD) contrast is widely used for probing brain activity, but its relationship to underlying neural activity remains elusive. Here, we combined fMRI with fiber-optic recordings of fluorescent calcium indicator signals to investigate this relationship in rat somatosensory cortex. Electrical forepaw stimulation (1-10 Hz) evoked fast calcium signals of neuronal origin that showed frequency-dependent adaptation. Additionally, slower calcium signals occurred in astrocyte networks, as verified by astrocyte-specific staining and two-photon microscopy. Without apparent glia activation, we could predict BOLD responses well from simultaneously recorded fiber-optic signals, assuming an impulse response function and taking into account neuronal adaptation. In cases with glia activation, we uncovered additional prolonged BOLD signal components. Our findings highlight the complexity of fMRI BOLD signals, involving both neuronal and glial activity. Combined fMRI and fiber-optic recordings should help to clarify cellular mechanisms underlying BOLD signals.

post to: CiteULike

Automated whole-cell patch-clamp electrophysiology of neurons in vivo.

Publication Date: 2012 May 6 PMID: 22561988
Authors: Kodandaramaiah, S. B. - Franzesi, G. T. - Chow, B. Y. - Boyden, E. S. - Forest, C. R.
Journal: Nat Methods

Whole-cell patch-clamp electrophysiology of neurons is a gold-standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput and quality of automated intracellular recording in mouse cortex and hippocampus.

post to: CiteULike

FMT-XCT: in vivo animal studies with hybrid fluorescence molecular tomography-X-ray computed tomography.

Publication Date: 2012 May 6 PMID: 22561987
Authors: Ale, A. - Ermolayev, V. - Herzog, E. - Cohrs, C. - de Angelis, M. H. - Ntziachristos, V.
Journal: Nat Methods

The development of hybrid optical tomography methods to improve imaging performance has been suggested over a decade ago and has been experimentally demonstrated in animals and humans. Here we examined in vivo performance of a camera-based hybrid fluorescence molecular tomography (FMT) system for 360 degrees imaging combined with X-ray computed tomography (XCT). Offering an accurately co-registered, information-rich hybrid data set, FMT-XCT has new imaging possibilities compared to stand-alone FMT and XCT. We applied FMT-XCT to a subcutaneous 4T1 tumor mouse model, an Aga2 osteogenesis imperfecta model and a Kras lung cancer mouse model, using XCT information during FMT inversion. We validated in vivo imaging results against post-mortem planar fluorescence images of cryoslices and histology data. Besides offering concurrent anatomical and functional information, FMT-XCT resulted in the most accurate FMT performance to date. These findings indicate that addition of FMT optics into the XCT gantry may be a potent upgrade for small-animal XCT systems.

post to: CiteULike

The 1000 Genomes Project: data management and community access.

Publication Date: 2012 PMID: 22543379
Authors: Clarke, L. - Zheng-Bradley, X. - Smith, R. - Kulesha, E. - Xiao, C. - Toneva, I. - Vaughan, B. - Preuss, D. - Leinonen, R. - Shumway, M. - Sherry, S. - Flicek, P.
Journal: Nat Methods

The 1000 Genomes Project was launched as one of the largest distributed data collection and analysis projects ever undertaken in biology. In addition to the primary scientific goals of creating both a deep catalog of human genetic variation and extensive methods to accurately discover and characterize variation using new sequencing technologies, the project makes all of its data publicly available. Members of the project data coordination center have developed and deployed several tools to enable widespread data access.

post to: CiteULike

Toward objective evaluation of proteomic algorithms.

Publication Date: 2012 PMID: 22543378
Authors: Yates, J. R. 3rd - Park, S. K. - Delahunty, C. M. - Xu, T. - Savas, J. N. - Cociorva, D. - Carvalho, P. C.
Journal: Nat Methods

post to: CiteULike

Close encounters: integrating nanopores and CMOS amplifiers for single-molecule detection.

Publication Date: 2012 PMID: 22543377
Authors: Oliver, J. S. - Dimitrov, V.
Journal: Nat Methods

post to: CiteULike

Hitting the sweet spot.

Publication Date: 2012 PMID: 22543376
Authors: Kulkarni, G. - Wadsworth, W. G.
Journal: Nat Methods

post to: CiteULike

Reprogramming in suspension.

Publication Date: 2012 PMID: 22543375
Authors: Chen, J. - Pei, D.
Journal: Nat Methods

post to: CiteULike

Direct protein control.

Publication Date: 2012 PMID: 22543374
Authors: Baker, M.
Journal: Nat Methods

post to: CiteULike

Enhanced photostability of cyanine fluorophores across the visible spectrum.

Publication Date: 2012 PMID: 22543373
Authors: Altman, R. B. - Zheng, Q. - Zhou, Z. - Terry, D. S. - Warren, J. D. - Blanchard, S. C.
Journal: Nat Methods

post to: CiteULike

Reply to "'Self-healing' dyes: intramolecular stabilization of organic fluorophores".

Publication Date: 2012 PMID: 22543372
Authors: Blanchard, S. C.
Journal: Nat Methods

post to: CiteULike

'Self-healing' dyes: intramolecular stabilization of organic fluorophores.

Publication Date: 2012 PMID: 22543371
Authors: Tinnefeld, P. - Cordes, T.
Journal: Nat Methods

post to: CiteULike

Fast, accurate error-correction of amplicon pyrosequences using Acacia.

Publication Date: 2012 PMID: 22543370
Authors: Bragg, L. - Stone, G. - Imelfort, M. - Hugenholtz, P. - Tyson, G. W.
Journal: Nat Methods

post to: CiteULike

An optimized two-finger archive for ZFN-mediated gene targeting.

Publication Date: 2012 Apr 29 PMID: 22543349
Authors: Gupta, A. - Christensen, R. G. - Rayla, A. L. - Lakshmanan, A. - Stormo, G. D. - Wolfe, S. A.
Journal: Nat Methods

The widespread use of zinc-finger nucleases (ZFNs) for genome engineering is hampered by the fact that only a subset of sequences can be efficiently recognized using published finger archives. We describe a set of validated two-finger modules that complement existing finger archives and expand the range of ZFN-accessible sequences threefold. Using this archive, we introduced lesions at 9 of 11 target sites in the zebrafish genome.

post to: CiteULike

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.

Publication Date: 2012 Apr 29 PMID: 22543348
Authors: Ries, J. - Kaplan, C. - Platonova, E. - Eghlidi, H. - Ewers, H.
Journal: Nat Methods

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.

post to: CiteULike

Faster STORM using compressed sensing.

Publication Date: 2012 Apr 22 PMID: 22522657
Authors: Zhu, L. - Zhang, W. - Elnatan, D. - Huang, B.
Journal: Nat Methods

In super-resolution microscopy methods based on single-molecule switching, the rate of accumulating single-molecule activation events often limits the time resolution. Here we developed a sparse-signal recovery technique using compressed sensing to analyze images with highly overlapping fluorescent spots. This method allows an activated fluorophore density an order of magnitude higher than what conventional single-molecule fitting methods can handle. Using this method, we demonstrated imaging microtubule dynamics in living cells with a time resolution of 3 s.

post to: CiteULike

An image analysis toolbox for high-throughput C. elegans assays.

Publication Date: 2012 Apr 22 PMID: 22522656
Authors: Wahlby, C. - Kamentsky, L. - Liu, Z. H. - Riklin-Raviv, T. - Conery, A. L. - O'Rourke, E. J. - Sokolnicki, K. L. - Visvikis, O. - Ljosa, V. - Irazoqui, J. E. - Golland, P. - Ruvkun, G. - Ausubel, F. M. - Carpenter, A. E.
Journal: Nat Methods

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available through the open-source CellProfiler project and enables objective scoring of whole-worm high-throughput image-based assays of C. elegans for the study of diverse biological pathways that are relevant to human disease.

post to: CiteULike

Systematic evaluation of factors influencing ChIP-seq fidelity.

Publication Date: 2012 Apr 22 PMID: 22522655
Authors: Chen, Y. - Negre, N. - Li, Q. - Mieczkowska, J. O. - Slattery, M. - Liu, T. - Zhang, Y. - Kim, T. K. - He, H. H. - Zieba, J. - Ruan, Y. - Bickel, P. J. - Myers, R. M. - Wold, B. J. - White, K. P. - Lieb, J. D. - Liu, X. S.
Journal: Nat Methods

We evaluated how variations in sequencing depth and other parameters influence interpretation of chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Using Drosophila melanogaster S2 cells, we generated ChIP-seq data sets for a site-specific transcription factor (Suppressor of Hairy-wing) and a histone modification (H3K36me3). We detected a chromatin-state bias: open chromatin regions yielded higher coverage, which led to false positives if not corrected. This bias had a greater effect on detection specificity than any base-composition bias. Paired-end sequencing revealed that single-end data underestimated ChIP-library complexity at high coverage. Removal of reads originating at the same base reduced false-positives but had little effect on detection sensitivity. Even at mappable-genome coverage depth of approximately 1 read per base pair, approximately 1% of the narrow peaks detected on a tiling array were missed by ChIP-seq. Evaluation of widely used ChIP-seq analysis tools suggests that adjustments or algorithm improvements are required to handle data sets with deep coverage.

post to: CiteULike