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Microscope imaging performance can be seriously degraded by optical inhomogeneities in biological samples. An adaptive optics approach using a spatial light modulator to divide the illumination wavefront into individually controllable subregions recovers near-diffraction–limited two-photon imaging performance in brain tissue.
By combining a protein complementation assay with a transcriptional reporter assay based on short expressed oligonucleotide tags (EXTs), the authors monitor tyrosine kinase receptor dimerization in conjunction with effector recruitment and downstream signaling.
A polarity-sensitive annexin-based biosensor called pSIVA becomes strongly fluorescent only after reversibly binding to the plasma membrane. pSIVA allows live-cell imaging of the apoptotic process in degenerating neurons in vitro and in vivo.
Staining with a mitochondrial dye permits high-purity isolation of cardiomyocytes from embryonic and induced pluripotent stem cells of several species, without genetic modification.
A degradation pathway found in plants, dependent on the hormone auxin, can be transplanted and harnessed to induce rapid and reversible target protein degradation in both yeast and animal cells.
Microsources positioned with holographic optical tweezers can establish a highly localized, three-dimensional chemical gradient that allows the manipulation of polarization and migration in single cells.
Tissue-specific expression of microRNA sponges allows precise regulation of microRNA activity in living flies. The authors investigate the role of miR-8 in the formation of neuromuscular junctions in detail.
Methods for automated fluorescence imaging allow high-throughput examination of reporter expression patterns in zebrafish embryos. They are applied to mapping promoter-enhancer interactions in this organism.
An improved version of the GCaMP genetically encoded calcium indicator, called GCaMP3, has higher calcium affinity and increased baseline fluorescence, dynamic range and stability. GCaMP3 performs better than existing genetically encoded calcium indicators in several assays and organisms, including in vivo imaging of neuronal signaling in worms, flies and mice.
Neuronal stimulation with channelrhodopsin-2 is combined with calcium fluorescence imaging to study neural connections in intact Caenorhabditis elegans.
Fusion of the genetically-encoded calcium indicator GCaMP2 to synaptophysin localizes the sensor to neuron presynaptic terminals and conveys linear responsiveness over a wider range of spike frequencies. The sensor allowed measurement of synaptic activity caused by spiking as well as graded voltage signals during in vivo imaging in zebrafish.
A combination of scattering interferometry and single-molecule fluorescence microscopy allows visualization of both the position and orientation of single Simian virus 40 particles on lipid bilayers and provides evidence of viral interaction with receptors in membrane nanodomains.
A combination of forward and reverse two hybrid screening allows systematic identification of 'edgetic' or edge-specific alleles, which encode proteins that have lost a single physical interaction but for which other interactions remain unperturbed.
Protein complexes can be detected, counted and localized within the bacterium Leptospira interrogans by combining quantitative mass spectrometry–based proteomics analysis with cryo-electron tomography, with the aid of an improved template-matching method.
Chromatin conformation capture on chip, or 4C, a technique developed to investigate the interaction of one chromosomal region with the rest of the chromatin, can also provide high resolution mapping of translocations and inversions in selected chromosomal regions.
Engineered splicing factors, consisting of an RNA recognition motif and a functional splicing module, can target a specific mRNA sequence and activate or suppress splicing of endogenous mRNAs.
Optically trapping an individual E. coli cell allows the long-term quantification of bacterial swimming phenotype: the stochastic transitions between 'running' and 'tumbling' as well as changes in swimming speed and direction.
Computational compensation for the loss of information from a cellular marker visualized in one fluorescence channel increases the number of markers that can be used to study a population of cells. This should allow a more detailed molecular understanding of heterogeneity in a cellular population.
High-throughput sequencing of Mariner transposon insertion libraries is used for quantitative studies of fitness and of genetic interactions in Streptococcus pneumoniae. The approach should allow similar studies in several microorganismal species.
By targeting a mutant Flp recombinase that forms a covalent protein-DNA complex to a single FRT site placed anywhere in the yeast genome, the authors can study repair pathways activated by a single genomic insult as well as events at the site of damage.