Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images from which phase contrast images can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be easily integrated with fluorescence microscopy.     

Gas phase infrared spectra via the phase integration quasi-classical method

We apply the recently developed phase integration method (PIM) (Monteferrante et al. Mol Phys. 2011;109:3015–3027) to the calculation of infrared spectra of gas phase molecules. The PIM combines a generalised Monte Carlo sampling of the exact thermal density of the system with classical molecular dynamics to obtain approximate time quantum correlation functions. To describe the molecules, we adopt very simple analytical potentials that have, however, proved interesting, and surprisingly challenging, benchmarks for approximate quantum dynamical schemes. We show that, in contrast with two other commonly applied methods, our spectra do not exhibit spurious features or unphysical shifts depending on the temperature. Identifying the positions of the peaks requires only a few tens of trajectories, while an accurate evaluation of the relative intensities of the peaks is computationally more demanding.     

Seamlessly expanding a randomized phase II trial to phase III

A sequential Bayesian phase II/III design is proposed for comparative clinical trials. The design is based on both survival time and discrete early events that may be related to survival and assumes a parametric mixture model. Phase II involves a small number of centers. Patients are randomized between treatments throughout, and sequential decisions are based on predictive probabilities of concluding superiority of the experimental treatment. Whether to stop early, continue, or shift into phase III is assessed repeatedly in phase II. Phase III begins when additional institutions are incorporated into the ongoing phase II trial. Simulation studies in the context of a non-small-cell lung cancer trial indicate that the proposed method maintains overall size and power while usually requiring substantially smaller sample size and shorter trial duration when compared with conventional group-sequential phase III designs.     

Temperature dependence of phase response curves for drug-induced phase shifts

The effectiveness of drugs active in phase-shifting the circadian rhythm of bioluminescent glow in the unicellular dinoflagellate Gonyaulax polyedra differs, depending upon the time of drug exposure (as pulses). For two drugs tested--cycloheximide and anisomycin, both inhibitors of cytosolic protein synthesis--this function, referred to as the drug phase response curve (dPRC), differs, depending upon the ambient temperature. Since dPRCs may differ at different drug concentrations, the effects observed may be attributable to differences in the effectiveness of or recovery from the drugs at different temperatures.     

Tomographic phase microscopy

We report a technique for quantitative three-dimensional (3D) mapping of refractive index in live cells and tissues using a phase-shifting laser interferometric microscope with variable illumination angle. We demonstrate tomographic imaging of cells and multicellular organisms, and time-dependent changes in cell structure. Our results will permit quantitative characterization of specimen-induced aberrations in high-resolution microscopy and have multiple applications in tissue light scattering.     

It's just a phase:NLRP6 phase separations drive signaling

NLRP6 is a key innate immune sensor whose functions have remained understudied.In a recent Cell paper,Shen et al.evaluate the mechanism of NLRP6 activation and ...     

Effect of normalization and phase angle calculations on continuous relative phase

The purpose of this investigation was to determine if phase plot normalization and phase angle definitions would have an affect on continuous relative phase calculations. A subject ran on a treadmill while sagittal plane kinematic data were collected with a high-speed (180 Hz) camera. Segmental angular displacements and velocities were used to create phase plots, and examine the coordination between the leg and thigh. Continuous relative phase was calculated with a combination of two different amplitude normalization techniques, and two different phase angle definitions. Differences between the techniques were noted with a root mean square (RMS) calculation. RMS values indicated that there were differences in the configuration of the non-normalized and normalized continuous relative phase curves. Graphically and numerically, it was noted that normalization tended to modify the continuous relative phase curve configuration. Differences in continuous relative phase curves were due to a loss in the aspect ratio of the phase plot during normalization. Normalization tended to neglect the nonlinear forces acting on the system since it did not maintain the aspect ratio of the phase plot. Normalization is not necessary because the arc tangent function accounts for differences in amplitudes between the segments. RMS values indicated that there were profound differences in the continuous relative phase curve when the phase angle was normalized and a phase angle was calculated relative to the right horizontal axis.     

Osmotic stress induces a phase transition from interdigitated gel phase to bilayer gel phase in multilamellar vesicles of dihexadecylphosphatidylcholine

We have investigated the effects of poly(ethylene glycol) (PEG) on the structure and phase behavior of multilamellar vesicles of dihexadecylphosphatidylcholine (DHPC-MLVs) using an X-ray diffraction method. At low concentrations of PEG-6K (MW = 7500), DHPC-MLVs were in an interdigitated gel (L(beta)I) phase, a gel phase with interdigitated hydrocarbon chains. At around 24% (w/v) PEG 6K, a phase transition from the L(beta)I phase to a bilayer gel phase occurred in the DHPC-MLVs, and above this concentration, they were in a bilayer gel phase. On the other hand, ethylene glycol (EG), the monomer of PEG, did not induce this phase transition in the DHPC-MLVs. A mechanism of this phase transition is proposed and discussed; a decrease in the repulsive interaction between the head groups of the phospholipids in the bilayer gel phase with an increase in PEG concentration, which is due to a decrease in the cross-sectional area of the head group region by osmotic stress, may be the main reason for this phase transition.     

Conversion of the beckman liquid phase sequencer to a gas-liquid phase sequencer

As an effective aid to extend the microsequencing capabilities the Beckman protein/peptide sequenator Series 890C has been successfully converted to a gas-liquid system, in which coupling buffer 25% trimethylamine was employed as a gas, and heptafluorobutyric acid as a liquid. The system has been found to be efficient for microsequencing (less than 100 pmol). The details of mechanical, plumbing, and other minor changes are described in this paper along with the results of sequencing proteins and peptides, directly and from blots.     

Face to phase: pitfalls in time delay estimation from coherency phase

Coherency phase is often interpreted as a time delay reflecting a transmission delay between spatially separated neural populations. However, time delays estimated from corticomuscular coherency are conflicting and often shorter than expected physiologically. Recent work suggests that corticomuscular coherence is influenced by afferent sensory feedback and bidirectional interactions. We investigated how bidirectional interaction affects time delay estimated from coherency, using a feedback model of the corticomuscular system. We also evaluated the effect of bidirectional interaction on two popular directed connectivity measures: directed transfer function (DTF) and partial directed coherence (PDC). The model is able to reproduce the range of time delays found experimentally from coherency phase by varying the strengths of the efferent and afferent pathways and the recording of sensory feedback in the cortical signal. Both coherency phase and DTF phase were affected by sensory feedback, resulting in an underestimation of the transmission delay. Coherency phase was altered by the recording of sensory feedback in the cortical signals and both measures were affected by the presence of a closed loop feedback system. Only PDC phase led to the correct estimation of efferent transmission delay in all simulated model configurations. Coherency and DTF phase should not be used to estimate transmission delays in neural networks as the estimated time delays are meaningless in the presence of sensory feedback and closed feedback loops.     

Defective S phase chromatin assembly causes DNA damage,activation of the S phase checkpoint,and S phase arrest

The S phase checkpoint protects the genome from spontaneous damage during DNA replication, although the cause of damage has been unknown. We used a dominant-negative mutant of a subunit of CAF-I, a complex that assembles newly synthesized DNA into nucleosomes, to inhibit S phase chromatin assembly and found that this induced S phase arrest. Arrest was accompanied by DNA damage and S phase checkpoint activation and required ATR or ATM kinase activity. These results show that in human cells CAF-I activity is required for completion of S phase and that a defect in chromatin assembly can itself induce DNA damage. We propose that errors in chromatin assembly, occurring spontaneously or caused by genetic mutations or environmental agents, contribute to genome instability.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer–induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10–15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.     

Biomolecules at phase boundaries

Experimental and theoretical studies of biomolecules at water surfaces and metal surfaces are presented. We studied lecithin molecules (monolayers) and phospholipase A₂ at a water surface with molecular dynamics (MD) simulations. Results were compared with those obtained for a pure water surface. We also studied amino acids at a TiO₂ surface with thermal desorption spectroscopy in the presence and absence of water.     

Isothermal lipid phase transitions

In liotropic lipid systems phase transitions can be induced isothermally by changing the solvent concentration or composition; alternatively, lipid composition can be modified by (bio)chemical means. The probability for isothermal phase transitions increases with the decreasing transition entropy; it is proportional to the magnitude of the transition temperature shift caused by transformation-inducing system variation. Manipulations causing large thermodynamic effects, such as lipid (de)hydration, binding of protons or divalent ions and macromolecular adsorption, but also close bilayer approach are, therefore, likely to cause structural lipid change(s) at a constant temperature. Net lipid charges enhance the membrane susceptibility to salt-induced isothermal phase transitions; a large proportion of this effect is due to the bilayer dehydration, however, rather than being a consequence of the decreased Coulombic electrostatic interactions. Membrane propensity for isothermal phase transitions, consequently, always increases with the hydrophilicity of the lipid heads, as well as with the desaturation and shortening of the lipid chains. Upon a phase change at a constant temperature, some of the interfacially bound solutes (e.g. protons or calcium) are released in the solution. Membrane permeability and fusogenicity simultaneously increase. In mixed systems, isothermal phase transitions, moreover, may result in lateral phase separation. All this opens up ways for the involvement of isothermal phase transitions in the regulation of biological processes.     

Texturising by phase separation

The most common food processing operations, thermal and mechanical treatments, both affect phase state and texture of foods. The phase separation threshold for food biopolymer mixtures is usually below their concentrations characteristically found in food. Phase-separation underpins texturisation processes during food processing and digestion. The distribution of water between the phases, the adsorption of lipids between the phases, the deformation of dispersed particles in flowing water-in-water emulsions and the gelation of the phases result in the specific morphology and texture of food. A historical experience in texturisation includes the development of cooked food and texturized vegetable analogues of animal foods.