Nature Methods Contents: April 2017 Volume 14 pp 325 - 456

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Advertisement Go Green! Try Non-toxic Nucleic Acid Gel Stains Environmental Friendly Replace Ethidium Bromide (EtBr) More Sensitive Learn more >> TABLE OF CONTENTS April 2017 Volume 14, Issue 4 In This Issue Special Feature Editorial This Month Correspondence Research Highlights Technology Feature News and Views Reviews Perspective Analysis Brief Communications Articles Advertisement   Say goodbye to sore thumbs with a $3,000 pipetting robot! Join the hundreds of labs, including Stanford, Harvard, and Mayo Clinic using Opentrons liquid-handling robots for workflow automation. It is as easy as downloading a protocol, loading your deck, and hitting run.  Click here to learn more about our personal pipetting robots, starting at $3,000. Subscribe   Facebook   RSS   Recommend to library   Twitter   Advertisement CALL FOR ENTRIES! Now is the time to enter Nikon's Small World Photo and Video Competitions. April 30th is the deadline to submit your images and movies taken through the light microscope. In celebration of Nikon's 100th anniversary, the grand prizewinners will receive $3000 and a trip to Japan. The lucky winners will also receive a private tour of Nikon's glass and lens manufacturing facilities. For more info, visit enter.nikonsw.com   Advertisement Nature Index Japan 2017 Japan's status as a science superstar is vulnerable. Nature Index 2017 Japan reveals that although the country is still among the upper echelons of global research, its output has continued to slide. Read the full supplement free for six months     Advertisement Looking to recruit? Limited time offer from Naturejobs:  Get a 20% discount on our Premium job posting package when you buy before March 31, 2017. Reach a potential audience of 10.8 million users* via nature.com Be seen first by candidates — your position will appear at the top of related searches for 60 days and be displayed prominently on the Naturejobs home page as Job of the Week. Plus appear as 'Featured' in search results Post a job now. * Publisher data 2016     In This Issue Top In This Issue      Special Feature Top Focus on deep imaging of live tissue Contents Editorial This Month Reviews Perspective Brief Communication Article Collection One of the goals of biological imaging is to watch biological processes where they occur – within living tissue or even within a living animal. But in vivo imaging presents a set of challenges that are not encountered when imaging relatively small, flat samples like cells. In this Focus issue, we bring together papers on methods for optical imaging within living tissue. Two Reviews discuss: microscopy methods for functional brain imaging and the principles and practicalities of the different flavours of light sheet microscopy. A Perspective describes the use of adaptive optics to correct aberrations in scattering samples. This issue also includes primary research papers on multiphoton microscopy, an editorial that reminds our readers of the many areas of this large and exciting field that we do not cover, and a collection of recent papers on deep imaging from Nature Research journals.   Editorial Top Focus on deep imaging of live tissue Imaging beyond the coverslip   p325 doi:10.1038/nmeth.4255   This Month Top Focus on deep imaging of live tissue The Author File: Chris Xu   p327 Vivien Marx doi:10.1038/nmeth.4231 Deep brain imaging with three-photon microscopy and a swim from the US to China. Points of Significance: Tabular data   pp329 - 330 Naomi Altman and Martin Krzywinski doi:10.1038/nmeth.4239 Tabulating the number of objects in categories of interest dates back to the earliest records of commerce and population censuses.   Correspondence Top MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy   pp331 - 332 Shawn Q Zheng, Eugene Palovcak, Jean-Paul Armache, Kliment A Verba, Yifan Cheng et al. doi:10.1038/nmeth.4193 MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions. Automatic tracing of ultra-volumes of neuronal images   pp332 - 333 Hanchuan Peng, Zhi Zhou, Erik Meijering, Ting Zhao, Giorgio A Ascoli et al. doi:10.1038/nmeth.4233 Automated tracing algorithms can extract neuronal morphology from fluorescent or brightfield images. UltraTracer scales up the capability of existing tracing algorithms to handle datasets of ever-increasing size.   Research Highlights Top Connectomics at the single-cell level Trans-synaptic tracing that starts from a single neuron provides a detailed view of that neuron's inputs. A magnetic alternative to FRET A new approach measures nanoscale distances based on magnetic resonance tuning. SLICE-ing a genome's architecture Thin slices through nuclei provide an unbiased view of the complex 3D organization of mammalian genomes. Volumetric functional imaging two ways Bessel focus scanning and stereoscopy speed up volumetric brain imaging. DNA variants or DNA damage? Cryptic, widespread DNA damage is commonly interpreted as sequence variation. DNA variants or DNA damage? Cryptic, widespread DNA damage is commonly interpreted as sequence variation. Methods in Brief STED with twice the depletion | Solution-state 13C NMR gets a boost | Optogenetic feedback in real time | Imaging fast subcellular dynamics with light sheets Tools in Brief A method for labeling methionines | One mouse to trap them all | Single-base editing | Genome scaffolding while you sequence Methods JOBS of the week Post-Doctoral Research Fellow, Statistical Methods Development Fred Hutchinson Cancer Research Center Experienced International Researcher LE STUDIUM LOIRE VALLEY INSTITUTE FOR ADVANCED STUDIES PostDoc Investigating Ladderane Lipid Biosynthesis Max Planck Institute for Medical Research Postdoctoral Fellow Vacancy - Interfacial Lab King Abdullah University of Science and Technology (KAUST) Senior Scientist Computational Toxicologist-Toxicokinetics Philip Morris International (PMI) More Science jobs from Methods EVENT NETTAB 2017 Methods, tools & platforms for Personalized Medicine in the Big Data Era 16.10.17 Palermo, Italy More science events from   Technology Feature Top Choosing CRISPR-based screens in cancer   pp343 - 346 Vivien Marx doi:10.1038/nmeth.4232 Many possibilities for parsing cancer emerge when labs combine gene editing and screens. And RNAi retains its spot in the menu of options.   News and Views Top Nanopore sequencing meets epigenetics   pp347 - 348 Michael C Schatz doi:10.1038/nmeth.4240 Advanced signal processing techniques enable the genome-wide analysis of methylation in native DNA using nanopore sequencing. See also: Brief Communication by Simpson et al. | Brief Communication by Rand et al.   Reviews Top Focus on deep imaging of live tissue In vivo imaging of neural activity   pp349 - 359 Weijian Yang and Rafael Yuste doi:10.1038/nmeth.4230 Yang and Yuste review currently available technologies for optical imaging of neural circuits, comparing them to help researchers choose optimal ones for their applications. Focus on deep imaging of live tissue A guide to light-sheet fluorescence microscopy for multiscale imaging   pp360 - 373 Rory M Power and Jan Huisken doi:10.1038/nmeth.4224 This Review introduces the fundamental considerations for building a light sheet microscope, describes the pros and cons associated with available implementations, and offers practical advice for users.   Perspective Top Focus on deep imaging of live tissue Adaptive optical fluorescence microscopy   pp374 - 380 Na Ji doi:10.1038/nmeth.4218 This Perspective introduces the development and use of adaptive optics in correcting aberrations in deep optical imaging applications.   Analysis Top Power analysis of single-cell RNA-sequencing experiments   pp381 - 387 Valentine Svensson, Kedar Nath Natarajan, Lam-Ha Ly, Ricardo J Miragaia, Charlotte Labalette et al. doi:10.1038/nmeth.4220 A comparison framework applied to 15 single-cell RNA-seq protocols reveals differences in accuracy and sensitivity and discusses the utility of RNA spike-in standards.   Brief Communications Top Focus on deep imaging of live tissue In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain   pp388 - 390 Dimitre G Ouzounov, Tianyu Wang, Mengran Wang, Danielle D Feng, Nicholas G Horton et al. doi:10.1038/nmeth.4183 Ouzounov et al. report calcium imaging with three-photon microscopy in the mouse brain. The approach enabled noninvasive recording of activity with high spatial and temporal resolution from GCaMP6-labeled neurons located as deep as the hippocampus. Optogenetic control with a photocleavable protein, PhoCl   pp391 - 394 Wei Zhang, Alexander W Lohman, Yevgeniya Zhuravlova, Xiaocen Lu, Matthew D Wiens et al. doi:10.1038/nmeth.4222 A photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after illumination with violet light expands the toolkit for cellular optogenetics. Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput   pp395 - 398 Todd M Gierahn, Marc H Wadsworth II, Travis K Hughes, Bryan D Bryson, Andrew Butler et al. doi:10.1038/nmeth.4179 Seq-Well provides similar scale and data quality to massively parallel, droplet-based single-cell RNA-seq methods in an easy to use, inexpensive and portable microwell format compatible with low-input samples. Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED   pp399 - 402 M Jason de la Cruz, Johan Hattne, Dan Shi, Paul Seidler, Jose Rodriguez et al. doi:10.1038/nmeth.4178 Fragmentation of large, imperfect crystals into nanocrystals by sonication, vortexing, or vigorous pipetting facilitates atomic-resolution analysis by the cryo-EM method MicroED. Prospective identification of hematopoietic lineage choice by deep learning   pp403 - 406 Felix Buggenthin, Florian Buettner, Philipp S Hoppe, Max Endele, Manuel Kroiss et al. doi:10.1038/nmeth.4182 A deep learning approach enables fast and robust prediction of hematopoietic stem cell lineage choice in time-lapse imaging three generations before conventional marker onset. Detecting DNA cytosine methylation using nanopore sequencing   pp407 - 410 Jared T Simpson, Rachael E Workman, P C Zuzarte, Matei David, L J Dursi et al. doi:10.1038/nmeth.4184 A hidden Markov model (HMM)-based tool enables detection of 5-methylcytosine (5-mC) from single-molecule nanopore-sequencing data generated directly from human genomic DNA without chemical treatment. See also: News and Views by Schatz Mapping DNA methylation with high-throughput nanopore sequencing   pp411 - 413 Arthur C Rand, Miten Jain, Jordan M Eizenga, Audrey Musselman-Brown, Hugh E Olsen et al. doi:10.1038/nmeth.4189 A tool based on hidden Markov model and hierarchical Dirichlet process (HMM-HDP) can call two methylated cytosine variants and a methylated adenine variant directly from genomic DNA using nanopore sequencing data. See also: News and Views by Schatz Visualization and analysis of single-cell RNA-seq data by kernel-based similarity learning   pp414 - 416 Bo Wang, Junjie Zhu, Emma Pierson, Daniele Ramazzotti and Serafim Batzoglou doi:10.1038/nmeth.4207 The SIMLR software identifies similarities between cells across a range of single-cell RNA-seq data, enabling effective dimension reduction, clustering and visualization. Salmon provides fast and bias-aware quantification of transcript expression   pp417 - 419 Rob Patro, Geet Duggal, Michael I Love, Rafael A Irizarry and Carl Kingsford doi:10.1038/nmeth.4197 Salmon is a computational tool that uses sample-specific models and a dual-phase inference procedure to correct biases in RNA-seq data and rapidly quantify transcript abundances. Advertisement Validated Western Blotting Antibodies That Work Every Time  Finding a western blotting antibody that works no longer requires trial and error - PrecisionAb™ Western Blotting Antibodies are validated using the industry's most stringent validation protocol and guaranteed to work in every lab. > Explore PrecisionAb Antibodies      Articles Top Focus on deep imaging of live tissue Volumetric two-photon imaging of neurons using stereoscopy (vTwINS)   pp420 - 426 Alexander Song, Adam S Charles, Sue Ann Koay, Jeff L Gauthier, Stephan Y Thiberge et al. doi:10.1038/nmeth.4226 vTwINS enables high-speed volumetric calcium imaging via a V-shaped point spread function and a dedicated data-processing algorithm. Song et al. apply this strategy to image population activity in the mouse visual cortex and hippocampus. Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution   pp427 - 434 Gary C H Mo, Brian Ross, Fabian Hertel, Premashis Manna, Xinxing Yang et al. doi:10.1038/nmeth.4221 New fluorescent biosensors enable the first super-resolution imaging of enzyme activity in live cells via fluorescence fluctuation increase by contact (FLINC). Automated synaptic connectivity inference for volume electron microscopy   pp435 - 442 Sven Dorkenwald, Philipp J Schubert, Marius F Killinger, Gregor Urban, Shawn Mikula et al. doi:10.1038/nmeth.4206 SyConn is a computational framework that infers the synaptic wiring of neurons in volume electron microscopy data sets with machine learning. It has been applied to zebra finch, mouse and zebrafish neuronal tissue samples. Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers   pp443 - 449 Franklin D Fuller, Sheraz Gul, Ruchira Chatterjee, E Sethe Burgie, Iris D Young et al. doi:10.1038/nmeth.4195 A robust acoustic droplet ejection-drop-on-tape method delivers samples to an X-ray free-electron laser source for combined serial femtosecond crystallography and X-ray emission spectroscopy analysis, providing detailed insights into macromolecular reaction dynamics. A genetic system to study Plasmodium falciparum protein function   pp450 - 456 Jakob Birnbaum, Sven Flemming, Nick Reichard, Alexandra Blancke Soares, Paolo Mesén-Ramírez et al. doi:10.1038/nmeth.4223 Selection-linked integration in the Plasmodium genome allows for rapid selection of genes inserted at a genomic locus or induction of the inactivation of gene products. Advertisement Nature Protocols: Poster on the production of Chimeric Antigen Receptor T-cells This poster overviews the production of (CAR) T-cells, discussing the significant and unique challenges of the production process  Available to download free Produced with support from STEMCELL Technologies   Top Advertisement nature.com webcasts Springer Nature presents a custom webcast on: Evaluation of a novel microRNA whole transcriptome assay Date: Wednesday, April 12, 2017 Time: 9AM PDT |12PM EDT | 5PM BST | 6PM CEST Register for FREE Sponsored by: HTG Molecular Diagnostics   Natureevents is a fully searchable, multi-disciplinary database designed to maximise exposure for events organisers. The contents of the Natureevents Directory are now live. The digital version is available here. Find the latest scientific conferences, courses, meetings and symposia on natureevents.com. 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