2002-2003 Third Annual Mozart Birthday Concert

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Single Molecule Fluorescence Image Patterns Linked to Dipole Orientation and Axial Position: Application to Myosin Cross-Bridges in Muscle Fibers

Photoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.Fluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ(2) minimization.Results indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation

Field assessment of dog as sentinel animal for plague in endemic foci of Madagascar

Funding Information: Sincere thanks to Mrs. L Angeltine Ralafiarisoa for technical assistance and the staff of the Plague Unit for their assistance during sample collections. This work was funded by an internal research grant (Ref: PA 14.25) from the Institut Pasteur de Madagascar. This research was also funded in part by the Wellcome Trust [095171/Z/10/Z]. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.Peer reviewedPublisher PD

Time-reversed adapted-perturbation (TRAP) optical focusing onto dynamic objects inside scattering media

The ability to steer and focus light inside scattering media has long been sought for a multitude of applications. At present, the only feasible strategy to form optical foci inside scattering media is to guide photons by using either implanted or virtual guide stars, which can be inconvenient and limits the potential applications. Here we report a scheme for focusing light inside scattering media by employing intrinsic dynamics as guide stars. By adaptively time-reversing the perturbed component of the scattered light, we show that it is possible to focus light to the origin of the perturbation. Using this approach, we demonstrate non-invasive dynamic light focusing onto moving targets and imaging of a time-variant object obscured by highly scattering media. Anticipated applications include imaging and photoablation of angiogenic vessels in tumours, as well as other biomedical uses

Modeling of longitudinal polytomous outcome from complex survey data - application to investigate an association between mental distress and non-malignant respiratory diseases

<p>Abstract</p> <p>Background</p> <p>The data from longitudinal complex surveys based on multi-stage sampling designs contain cross-sectional dependencies among units due to clustered nature of the data and within-subject dependencies due to repeated measurements. Special statistical methods are required to analyze longitudinal complex survey data.</p> <p>Methods</p> <p>Statistics Canada's longitudinal National Population Health Survey (NPHS) dataset from the first five cycles (1994/1995 to 2002/2003) was used to investigate the effects of demographic, social, life-style, and health-related factors on the longitudinal changes of mental distress scores among the NPHS participants who self-reported physician diagnosed respiratory diseases, specifically asthma and chronic bronchitis. The NPHS longitudinal sample includes 17,276 persons of all ages. In this report, participants 15 years and older (n = 14,713) were considered for statistical analysis. Mental distress, an ordinal outcome variable (categories: no/low, moderate, and high) was examined. Ordered logistic regression models based on the weighted generalized estimating equations approach were fitted to investigate the association between respiratory diseases and mental distress adjusting for other covariates of interest. Variance estimates of regression coefficients were computed by using bootstrap methods. The final model was used to predict the probabilities of prevalence of no/low, moderate or high mental distress scores.</p> <p>Results</p> <p>Accounting for design effects does not vary the significance of the coefficients of the model. Participants suffering with chronic bronchitis were significantly at a higher risk (OR_adj= 1.37; 95% CI: 1.12-1.66) of reporting high levels of mental distress compared to those who did not self-report chronic bronchitis. There was no significant association between asthma and mental distress. There was a significant interaction between sex and self-perceived general health status indicating a dose-response relationship. Among females, the risk of mental distress increases with increasing deteriorating (from excellent to very poor) self-perceived general health.</p> <p>Conclusions</p> <p>A positive association was observed between the physician diagnosed self-reported chronic bronchitis and an increased prevalence of mental distress when adjusted for important covariates. Variance estimates of regression coefficients obtained from the sandwich estimator (i.e. not accounting for design effects) were similar to bootstrap variance estimates (i.e. accounting for design effects). Even though these two sets of variance estimates are similar, it is more appropriate to use bootstrap variance estimates.</p

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging

mKikGR, a Monomeric Photoswitchable Fluorescent Protein

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal