Table of contents



Building core strength p599


The scientific community benefits from infrastructure that facilitates the dissemination of cutting-edge methods.


This Month

The Author File: Don Arnold p601


To the tune of a classical guitar, finding a way to watch learning as it happens.

Points of Significance: Classification evaluation pp603 - 604

Jake Lever, Martin Krzywinski & Naomi Altman


It is important to understand both what a classification metric expresses and what it hides.



Response to "Confidence intervals are no salvation from the alleged fickleness of the P value" p606

Lewis G Halsey, Douglas Curran-Everett & Gordon B Drummond


See also: Correspondence by van Helden | Correspondence by Huber

The democratization of cryo-EM pp607 - 608

David I Stuart, Sriram Subramaniam & Nicola G A Abrescia



Research Highlights

Cas9, the cellular genealogist p609

Genome editing marks cells in developing fish for lineaging studies.

Conditional nanobody tools pp610 - 611

Nanobodies that are stable only in the presence of their antigen can be harnessed to manipulate or detect antigen-expressing cells.

Making sense of NAD+ subcellular localization pp610 - 611

A fluorescent NAD+ biosensor targeted to specific cellular compartments detects local fluctuation in NAD+ concentration.

The human transient transcriptome p612

Fragmenting transcripts after 4-thiouridine incorporation allows the quantification of even short-lived noncoding RNA.

Luciferase gets deep and sensitive p615

A luciferin analog enables highly sensitive bioluminescent imaging from deep within biological tissue samples.

Methods in Brief

Tools in Brief


Technology Feature

Stem cells: a dish of neurons pp617 - 622

Vivien Marx


Labs can generate neurons from pluripotent stem cells to study basic biology and to model disease. Protocols are getting more robust, and labs add personal preferences.


News and Views

The quest for multiplexed spatially resolved transcriptional profiling pp623 - 624

Carolina Wählby


Crowded transcript signals are resolved by cross-correlation or by gel expansion microscopy.

See also: Brief Communication by Coskun & Cai | Article by Chen et al.

A glance at N6-methyladenosine in transcript isoforms pp624 - 625

Hailing Shi & Chuan He


A sequencing approach, m6A-LAIC-seq, globally quantifies the proportion of m6A-modified transcripts of a specific gene and the differential m6A modification levels among its isoforms.

See also: Article by Molinie et al.




A practical guide to photoacoustic tomography in the life sciences pp627 - 638

Lihong V Wang & Junjie Yao


Photoacoustic imaging (PAI) can bridge the gap between high resolution optical imaging and deep tissue imaging applications. This Review introduces PAI as well as various implementations for a range of biological applications.

Contrast agents for molecular photoacoustic imaging pp639 - 650

Judith Weber, Paul C Beard & Sarah E Bohndiek


This Review covers genetically encoded and exogenous contrast agents for photoacoustic imaging and offers guidance for choosing optimal probes for biological applications on the basis of photophysical properties, targeting and performance.



Recognizing millions of consistently unidentified spectra across hundreds of shotgun proteomics datasets pp651 - 656

Johannes Griss, Yasset Perez-Riverol, Steve Lewis, David L Tabb, José A Dianes, Noemi del-Toro, Marc Rurik, Mathias Walzer, Oliver Kohlbacher, Henning Hermjakob, Rui Wang & Juan Antonio Vizcaíno


A newly developed algorithm enabled clustering of all 256 million (66 million identified and 190 million unidentified) peptide MS/MS spectra available in the PRIDE Archive database, allowing the detection of millions of consistently unidentified spectra across different data sets, of which roughly 20% could be identified using multiple complementary analysis tools.


Brief Communications

Dense transcript profiling in single cells by image correlation decoding pp657 - 660

Ahmet F Coskun & Long Cai


Correlation fluorescent in situ hybridization (corrFISH) makes it possible to quantify abundant transcripts using sequential barcoded hybridization, despite high spot density in images.

See also: News and Views by Wählby

Varying label density allows artifact-free analysis of membrane-protein nanoclusters pp661 - 664

Florian Baumgart, Andreas M Arnold, Konrad Leskovar, Kaj Staszek, Martin Fölser, Julian Weghuber, Hannes Stockinger & Gerhard J Schütz


PALM and STORM are powerful methods for studying membrane-protein clustering. However, fluorophore blinking can lead to miscounting artifacts. A new method shows that varying label density works for artifact-free analysis of membrane-protein nanoclusters.



Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution pp665 - 672

Martin E Masip, Jan Huebinger, Jens Christmann, Ola Sabet, Frank Wehner, Antonios Konitsiotis, Günther R Fuhr & Philippe I H Bastiaens


Reversible cryo-arrest allows consecutive super-resolution and functional imaging of molecular patterns in the same mammalian cell. This method was used to study the evolution of receptor tyrosine kinase reaction patterns at multiple scales.

An E3-ligase-based method for ablating inhibitory synapses pp673 - 678

Garrett G Gross, Christoph Straub, Jimena Perez-Sanchez, William P Dempsey, Jason A Junge, Richard W Roberts, Le A Trinh, Scott E Fraser, Yves De Koninck, Paul De Koninck, Bernardo L Sabatini & Don B Arnold


When studying neural circuitry, the ablation of synapses may be an alternative to optogenetic manipulation of neurons. A genetically encoded tool called GFE3 eliminates inhibitory inputs into neurons expressing GFE3.

Nanoscale imaging of RNA with expansion microscopy pp679 - 684

Fei Chen, Asmamaw T Wassie, Allison J Cote, Anubhav Sinha, Shahar Alon, Shoh Asano, Evan R Daugharthy, Jae-Byum Chang, Adam Marblestone, George M Church, Arjun Raj & Edward S Boyden


ExFISH extends expansion microscopy to single-molecule RNA imaging, enabling super-resolution imaging of diverse RNAs in cells and tissues on conventional microscopes. The method enables multiplexed imaging of RNA and improved RNA quantitation.

See also: News and Views by Wählby

UMI-4C for quantitative and targeted chromosomal contact profiling pp685 - 691

Omer Schwartzman, Zohar Mukamel, Noa Oded-Elkayam, Pedro Olivares-Chauvet, Yaniv Lubling, Gilad Landan, Shai Izraeli & Amos Tanay


UMI-4C is a rapid, simplified barcoding approach to targeted chromatin conformation capture that produces high-complexity libraries from low sample input, is easily multiplexed and gives a quantitative, statistically defined readout.

m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome pp692 - 698

Benoit Molinie, Jinkai Wang, Kok Seong Lim, Roman Hillebrand, Zhi-xiang Lu, Nicholas Van Wittenberghe, Benjamin D Howard, Kaveh Daneshvar, Alan C Mullen, Peter Dedon, Yi Xing & Cosmas C Giallourakis


m6A-LAIC-seq quantifies the levels of N6-methyladenosine in isoforms of the same gene in a cell-type-specific manner.

See also: News and Views by Shi & He