Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

We present a single-molecule tool called the CoPro (Concentration of Proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrates its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a 4-fold shift towards higher values in concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, which is almost exclusively localized in the nucleus under high and..

Interactions with combined chemical cues inform harvester ant foragers' decisions to leave the nest in search of food.

Social insect colonies operate without central control or any global assessment of what needs to be done by workers. Colony organization arises from the responses of individuals to local cues. Red harvester ants (Pogonomyrmex barbatus) regulate foraging using interactions between returning and outgoing foragers. The rate at which foragers return with seeds, a measure of food availability, sets the rate at which outgoing foragers leave the nest on foraging trips. We used mimics to test whether outgoing foragers inside the nest respond to the odor of food, oleic acid, the odor of the forager itself, cuticular hydrocarbons, or a combination of both with increased foraging activity. We compared foraging activity, the rate at which foragers passed a line on a trail, before and after the addition of mimics. The combination of both odors, those of food and of foragers, is required to stimulate foraging. The addition of blank mimics, mimics coated with food odor alone, or mimics coated with forager odor alone did not increase foraging activity. We compared the rates at which foragers inside the nest interacted with other ants, blank mimics, and mimics coated with a combination of food and forager odor. Foragers inside the nest interacted more with mimics coated with combined forager/seed odors than with blank mimics, and these interactions had the same effect as those with other foragers. Outgoing foragers inside the nest entrance are stimulated to leave the nest in search of food by interacting with foragers returning with seeds. By using the combined odors of forager cuticular hydrocarbons and of seeds, the colony captures precise information, on the timescale of seconds, about the current availability of food

On the Josephson Coupling between a disk of one superconductor and a surrounding superconducting film of a different symmetry

A cylindrical Josephson junction with a spatially dependent Josephson coupling which averages to zero is studied in order to model the physics of a disk of d-wave superconductor embedded in a superconducting film of a different symmetry. It is found that the system always introduces Josepshon vortices in order to gain energy at the junction. The critical current is calculated. It is argued that a recent experiment claimed to provide evidence for s-wave superconductivity in $YBa_2Cu_3O_7$ may also be consistent with d-wave superconductivity. Figures available from the author on request.Comment: 10 pages, revtex3.0, TM-11111-940321-1

The use of multilayer network analysis in animal behaviour

Network analysis has driven key developments in research on animal behaviour by providing quantitative methods to study the social structures of animal groups and populations. A recent formalism, known as \emph{multilayer network analysis}, has advanced the study of multifaceted networked systems in many disciplines. It offers novel ways to study and quantify animal behaviour as connected 'layers' of interactions. In this article, we review common questions in animal behaviour that can be studied using a multilayer approach, and we link these questions to specific analyses. We outline the types of behavioural data and questions that may be suitable to study using multilayer network analysis. We detail several multilayer methods, which can provide new insights into questions about animal sociality at individual, group, population, and evolutionary levels of organisation. We give examples for how to implement multilayer methods to demonstrate how taking a multilayer approach can alter inferences about social structure and the positions of individuals within such a structure. Finally, we discuss caveats to undertaking multilayer network analysis in the study of animal social networks, and we call attention to methodological challenges for the application of these approaches. Our aim is to instigate the study of new questions about animal sociality using the new toolbox of multilayer network analysis.Comment: Thoroughly revised; title changed slightl

Test Experiment for Time-Reversal Symmetry Breaking Superconductivity

A new experiment is proposed to probe the time-reversal symmetry of a superconductor. It is shown that a time-reversal symmetry breaking superconductor can be identified by the observation of a fractional flux in connection with a Josephson junction in a special geometry.Comment: 4 pages, 2 figures available upon request, Revtex, MIT-CMT-OC

Observation and interpretation of motional sideband asymmetry in a quantum electro-mechanical device

Quantum electro-mechanical systems offer a unique opportunity to probe quantum noise properties in macroscopic devices, properties which ultimately stem from the Heisenberg Uncertainty Principle. A simple example of this is expected to occur in a microwave parametric transducer, where mechanical motion generates motional sidebands corresponding to the up and down frequency-conversion of microwave photons. Due to quantum vacuum noise, the rates of these processes are expected to be unequal. We measure this fundamental imbalance in a microwave transducer coupled to a radio-frequency mechanical mode, cooled near the ground state of motion. We also discuss the subtle origin of this imbalance: depending on the measurement scheme, the imbalance is most naturally attributed to the quantum fluctuations of either the mechanical mode or of the electromagnetic field

Quantum squeezing of motion in a mechanical resonator

As a result of the quantum, wave-like nature of the physical world, a harmonic oscillator can never be completely at rest. Even in the quantum ground state, its position will always have fluctuations, called the zero-point motion. Although the zero-point fluctuations are unavoidable, they can be manipulated. In this work, using microwave frequency radiation pressure, we both prepare a micron-scale mechanical system in a state near the quantum ground state and then manipulate its thermal fluctuations to produce a stationary, quadrature-squeezed state. We deduce that the variance of one motional quadrature is 0.80 times the zero-point level, or 1 dB of sub-zero-point squeezing. This work is relevant to the quantum engineering of states of matter at large length scales, the study of decoherence of large quantum systems, and for the realization of ultra-sensitive sensing of force and motion

A Mass-Loss Rate Determination For Zeta Puppis From The Quantitative Analysis Of X-Ray Emission-Line Profiles

We fit every emission line in the high-resolution Chandra grating spectrum of. Pup with an empirical line profile model that accounts for the effects of Doppler broadening and attenuation by the bulk wind. For each of 16 lines or line complexes that can be reliably measured, we determine a best-fitting fiducial optical depth, tau(*) equivalent to kappa(M) over dot/4 pi R(*)upsilon(infinity), and place confidence limits on this parameter. These 16 lines include seven that have not previously been reported on in the literature. The extended wavelength range of these lines allows us to infer, for the first time, a clear increase in tau(*) with line wavelength, as expected from the wavelength increase of bound-free absorption opacity. The small overall values of tau(*), reflected in the rather modest asymmetry in the line profiles, can moreover all be fitted simultaneously by simply assuming a moderate mass-loss rate of 3.5 +/- 0.3 x 10(-6) M(circle dot) yr(-1), without any need to invoke porosity effects in the wind. The quoted uncertainty is statistical, but the largest source of uncertainty in the derived mass-loss rate is due to the uncertainty in the elemental abundances of zeta Pup, which affects the continuum opacity of the wind, and which we estimate to be a factor of 2. Even so, the mass-loss rate we find is significantly below the most recent smooth-wind H alpha mass-loss rate determinations for zeta Pup, but is in line with newer determinations that account for small-scale wind clumping. If zeta Pup is representative of other massive stars, these results will have important implications for stellar and Galactic evolution