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Experimental and analytical methods are described for in vivo single-molecule imaging of GFP-tagged proteins at the cell surface and are applied to the developing C. elegans embryo.
Excitation using rotating polarized light and detection of periodic signals from rectangular nanoareas allows widef-ield super-resolution imaging of biological structures in cells and in tissue with reduced background.
Screening of cells transduced with a lentivirus library harboring nucleosome-free regions of mammalian genomes identifies cis elements that regulate transcription.
Putative enhancer elements of a genomic region of interest are cloned in front of a reporter gene, integrated in a single site in mouse embryonic stem cells, and screened for enhancer activity using flow cytometry and high-throughput sequencing.
Three new red-excitable monomeric fluorescent proteins obtained by structure-guided mutagenesis of mNeptune are described in this work. The authors show the use of one of them, mCardinal, to visualize the differentiation of myoblasts into myocytes in living mice.
Single-molecule structural transitions involving DNA twisting can be measured with substantially greater spatiotemporal resolution than previously possible with a gold rotor bead tracking (AuRBT) method. This approach uses magnetic tweezers and evanescent darkfield microscopy to track a gold nanoparticle probe attached to a DNA molecule.
This paper reports conditions for the in vitro culture of human leukemia stem cells, which should enable basic studies as well as chemical screens on these cells.
Cryo-scanning transmission electron tomography (CSTET) of unstained, fully hydrated vitrified biological specimens is shown to have advantages over cryo-electron tomography (CET), notably at high sample tilts providing greater depth resolution for thick samples.
High-throughput in vitro analysis of TALE nuclease (TALEN) cleavage specificity gives insight into the mechanism of TALE binding and allows the design of TALENs with lower off-target activity.
Sequencing the transcriptomes of more than 100 species of alga yields new channelrhodopsins with promising properties for optogenetics. A far red–shifted channelrhodopsin, Chrimson, opens up new behavioral capabilities in Drosophila, and alongside a fast yet light-sensitive blue channelrhodopsin, Chronos, enables independent excitation of two neuronal populations in brain slices.
A streamlined, robust sample-preparation method for mass spectrometry–based proteome analysis is reported. All sample preparation steps are carried out in a single enclosed reactor, reducing the potential for contamination and losses, and enabling comprehensive proteome coverage.
Variations on point accumulation for imaging in nanoscale topography (PAINT) for super-resolution imaging extend the technology to allow simple imaging of cellular proteins as well as synthetic DNA nanostructures and provide a high level of multiplexing.
Similarity network fusion (SNF) is an approach to integrate multiple data types on the basis of similarity between biological samples rather than individual measurements. The authors demonstrate SNF by constructing patient networks to identify disease subtypes with differential survival profiles.
A collection of improved FRET-based calcium biosensors, called Twitch sensors, is described. Twitches have a reduced number of calcium binding sites per sensor and display high sensitivity in in vivo imaging experiments in mouse brain and lymph node.