Research articles

The authors compare quality metrics of libraries from seven strand-specific RNA-seq methods in terms of complexity, strand specificity, evenness and continuity of coverage, and expression profiling. They provide a computational pipeline to compare these metrics from any RNA-seq protocol.

This resource provides a comprehensive evaluation of rodent locomotor profiles after different types of lesions to the central nervous system. The data set can guide the selection of suitable lesion paradigms, locomotor tasks and readouts in future animal studies.

The combination of protein display, moderate selection for protein activity and high-throughput DNA sequencing can be applied to hundreds of thousands of protein variants in parallel, enabling the derivation of sequence-function relationships.

Use of a trimethoprim chemical tag allows super-resolution live-cell microscopy by stochastic single molecule–based localization imaging of the dynamics of genetically tagged histone H2B in cell nuclei.

Engineering of the Ca²⁺-sensing domain in existing yellow Cameleon Ca²⁺ indicators is used to create indicators with a range of increased Ca²⁺ affinities capable of detecting subtle changes in intracellular Ca²⁺ at low resting levels.

Incorporation of time information into the annotation of distinct biological states in automated fluorescence time-lapse live-cell imaging of complex cellular dynamics reduces both classification noise and confusion between cell states with similar morphology. A computational framework for achieving this is implemented in the open-source software package CellCognition.

Two-photon excitation of a phosphorescent nanoprobe that is quenched by molecular oxygen permits high-resolution measurements of oxygen in both the vasculature and tissue of rodent brain.

A simple automated system to simultaneously measure growth rate and lag time of large numbers of bacteria permits the identification of subsets in a heterogenous population.

Micropost arrays can be used to modulate substrate rigidity independently of other substrate properties, permitting the study of the effects of rigidity on cell function.

A platform for rapid and automated imaging and laser manipulation of zebrafish larvae is presented. It should permit large-scale chemical and genetic screens in this vertebrate organism.

On-flowcell capture and reverse transcription followed by single-molecule cDNA sequencing provides reproducible digital gene expression results from as few as 1,000 cells.

Single integration of a target gene expressing MS2-binding stem loops allows the real time quantification of transcriptional bursts, promoter firings and cell cycle–dependent transcription rates.

Improving the protocols for chromatin immunoprecipitation and library construction for the Illumina Genome Analyzer allows for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) experiments on input samples as small as 10,000 cells and yields information on bivalent chromatin domains in hematopoietic progenitor cells.

Genetically encoded voltage-sensitive fluorescent proteins can be used to measure electrical activity from selected populations of neurons. This study demonstrates that these probes, when expressed in pyramidal cells of mouse somatosensory cortex, can report electrical responses in vivo. These proteins are a complementary tool to calcium imaging techniques for optical functional brain imaging.

A monomeric fluorescent protein that can be irreversibly photoswitched from green to red form, both of which can be reversibly photoactivated, is reported. It is applied to pulse-chase experiments in which dynamic structures in live cells are imaged with superresolution using photoactivation localization microscopy (PALM).

The combination of digital scanned laser light sheet microscopy and incoherent structured illumination allows intrinsic removal of scattered background fluorescence from the desired fluorescent signal. This provides substantial advantages for imaging nontransparent organisms and allowed reconstruction of a fly digital embryo from a developing Drosophila embryo.

A genetically encoded ratiometric biosensor not based on fluorescence resonance energy transfer (FRET), ClopHensor, allows concurrent measurement of intracellular pH and chloride by providing an internal control for pH-dependent fluorescence changes. Measurements of chloride levels in acidic large dense core vesicles showed high concentrations of chloride.

Multiphoton laser-scanning microscopy paired either with stationary line scans across a vessel or moving line scans across a network of vessels allows the profiling of key parameters that describe red blood cells.

Noncontact, frequency-modulation atomic force microscopy (FM-AFM) can be used to measure the microrheological properties of soft samples at acoustic frequencies. The method will be useful for characterizing the elasticity and viscosity of tissues that detect or produce sound.

Light-sensitive LOV domains show much promise for engineering proteins with photoswitchable activity. The dynamic range of a LOV domain is now substantially improved by the introduction of beneficial mutations predicted by an analytical model of photoswitching. The approach should prove useful to improve the function of multiple LOV-based switches.