Articles in 2010

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.

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.

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.

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.

Capturing the 5′ end of transcripts from an input of only 1,000 cells and linking the transcription start sites to downstream sequences allows the analysis of complex transcriptome architecture.

A set of computational and imaging approaches, called MARS-ALT, permits three-dimensional tracking of plant tissue development, including cell lineaging, at cellular resolution. It is applied to the study of floral development in Arabidopsis.

This technique allows functional imaging of neurons in head-fixed Drosophila while the fly walks on an air-supported ball. Using a genetically encoded calcium sensor, the activity of motion-sensitive neurons in the fly optic lobe was recorded while the flies were presented with visual stimuli. Activity in these cells correlated with robust optomotor behavior in the walking flies.

Expression of the transporter SID-1 in Caenorhabditis elegans neurons renders the cells sensitive to systemic RNAi and permits previously unidentified neuronal phenotypes to be uncovered. This expression also reduces RNAi in nonneuronal cell types, allowing examination of neuronal functions of lethal genes.

By micropatterning cells in polarized shapes the global distribution of endomembranes can be compared and subtle but statistically significant changes can be detected from only tens of cells.

This software integrates data from multiple samples and single-nucleotide polymorphism and comparative genome hybridization array platforms to detect and genotype copy-number variants (CNVs). It has high accuracy in the detection of short deletions and amplifications and will be valuable for genome-wide CNV analyses in population studies.

Paired-end reads consisting of 5′ transcription start sites and 3′ downstream sequences from transcripts in Drosophila melanogaster reveal distinct initiation patterns at different fly promoters and show that 5′ caps originating in coding regions are added posttranscriptionally.

A photoconvertible reporter of the ubiquitin-proteasome system permits detection of its activity independent of protein synthesis and is applied to study cell type– and age-specific protein degradation in living Caenorhabditis elegans.

Polymerase kinetics observed during single-molecule, real-time sequencing depend on the methylation status of the DNA template. Measurement of kinetic parameters such as interpulse duration and pulse width allows the identification of methylated adenosine in Escherichia coli and the distinction between 5-methylcytosine and 5-hydroxymethylcytosine in synthetic templates.

Combining reverse transfection of protein tyrosine kinase substrates on cell arrays with fluorescence resonance energy transfer (FRET) measurements by fluorescence lifetime imaging microscopy (FLIM) allows quantitative assessment of phosphorylation patterns and identification of feedback loops at single-cell resolution.

Limitations in scanning speed have made it difficult for two-photon imaging to provide accurate temporal information on neuronal signaling. Refinements to random-access scanning using acousto-optic deflectors and an automated algorithm for reconstructing complex spike trains allowed in vivo high-speed optical recording of spiking activity in neuronal populations in the mouse neocortex.

A conditional gene expression system in Caenorhabditis elegans is reported. It should permit the generation of temperature-sensitive alleles for most genes.

By stochastically sampling cells in groups of ten, the authors identify transcriptional programs with strong cell-to-cell expression differences thus allowing them to study endogenous heterogeneities in single cells.

Using a GFP complementation assay to tag effectors of the type-III secretion system in gram-negative bacteria allows localization of the effectors in the host cell in the course of bacterial infection.