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A combination of gradient refractive index lenses with plano-convex lenses produces high-resolution microlenses with image quality similar to a conventional high quality microscope objective. The microlenses are capable of imaging dendritic spines on hippocampal neurons in live mice.
Fluorescence resonance energy transfer (FRET) between a small-molecule fluorophore donor and a transition metal ion acceptor, a method called 'transition metal ion FRET,' works over shorter distances than the classical FRET approach and can thus be used to monitor very small conformational changes in proteins.
Using a topographically patterned substrate for immobilization of single yeast cells and a piezo-impact micromanipulator to transiently disrupt the cell wall, molecules can be physically introduced into yeast.
Activation of caged doxycycline or cyanodoxycycline by biologically innocuous doses of UV light allows for precise temporal and spatial control of transgene expression in hippocampal slices, mouse embryos and Xenopus laevis tadpoles.
Although fast temperature jump methods to study protein folding dynamics have long been applied, pressure has been a neglected thermodynamic parameter. A method to generate rapid and large drops in pressure is complementary to fast temperature jump methods and could be useful for direct comparisons to molecular dynamics simulations.
The protein interaction platform or PIP assay uses a viral scaffolding protein fused to a bait and a fluorescent reporter protein fused to putative prey as the basis for a simple visual screen for protein-protein interactions in yeast.
Genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura with an average insert size of 36 kilobases can easily be tagged and inserted into the fly genome. These resources will be valuable for evolutionary and developmental studies in the fly.
Two bacterial artificial chromosome (BAC) libraries, spanning almost the entire D. melanogaster genome in insert sizes of 20 and 80 kb, that allow easy integration into the fruit fly genome at defined docking sites provide a rich resource to study gene expression and function.
On-array synthesis of over 20,000 shRNAs at a coverage of ∼30 shRNAs per gene, followed by cloning into lentiviral shRNA libraries and deconvolution of the complex libraries by deep sequencing, ensures high confidence in the observed knockdown phenotypes with low false-negative rates and few off-target hits.
A multilaboratory analysis characterized the ability of 27 different labs to identify 20 proteins at equimolar concentrations in a highly purified test sample mixture using mass spectrometry. The results show that while the technology is reproducible, many common experimental problems arise, and improved search engines and databases are still needed.
Expressing uracil phosphoribosyltransferase in specific tissues in the fly allows the incorporation of 4-thiouracil into newly synthesized RNA in vivo. The thio-labeled RNA can then be isolated and analyzed by routine procedures allowing the cell type–specific measure of RNA synthesis and decay rates.
A modular, automatable system using recombineering to facilitate multigene assembly, called Acembl, provides a streamlined approach for expressing multiprotein complexes in Escherichia coli.
An automated system for tracking large numbers of fruit flies over time and for detecting their behaviors is presented, and should allow high-throughput quantitative studies of fly behavior.
As tissues mature, they undergo shape changes that are the result of individual and collective cell movement triggered by cell-autonomous behavior or external forces. By measuring patterns of strain rates the authors can model these forces and quantify tissue shaping behavior.
The use of a spatial light modulator for illuminating the sample in structured-illumination microscopy (SIM) increases imaging speed by three orders of magnitude. The resulting 100-nm resolution and 11-Hz frame rate allowed video imaging of tubulin polymerization and depolymerization as well as kinesin movement on microtubules.
Lentiviral vectors that express a fluorescent reporter and a selectable marker in pluripotent cells improve and simplify isolation of induced pluripotent stem cell lines in mouse and human.
A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts.
An atomic force microscope with a side-view fluorescent imaging path facilitates the direct correlation of mechanical force measurements with observations of changes in cell shape and cytoskeleton rearrangements resulting from the applied forces or during active generation of forces by the cell. The combined instrument could help lead to insights in understanding cell mechanics, contractility and cell-cell adhesion.
The combination of 5′ exonuclease, DNA polymerase and ligase with overlapping DNA fragments facilitates the in vitro assembly of large DNA constructs, including an entire bacterial genome.
Several red and orange fluorescent proteins are reported to be photoconvertible. Specifically, three red fluorescent proteins that can be switched to green, and two orange fluorescent proteins that can be switched to far red are reported.
