Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90–110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

Quantification of spread of cerebellar long-term depression with chemical two-photon uncaging of glutamate

Localized, chemical two-photon photolysis of caged glutamate was used to map the changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors caused by long-term synaptic depression (LTD) in cerebellar Purkinje cells. LTD produced by pairing parallel fiber activity with depolarization was accompanied by a decline in the response of Purkinje cells to uncaged glutamate that accounted for both the time course and magnitude of LTD. This depression of glutamate responses was observed not only at the site of parallel fiber stimulation but also at more distant sites. The amount of LTD decreased with distance and was half-maximal 50 μm away from the site of parallel fiber activity. Estimation of the number of parallel fibers active during LTD induction indicates that LTD modified glutamate receptors not only at active synapses but also at 600 times as many inactive synapses on a single Purkinje cell. Therefore, both active and inactive parallel fiber synapses can undergo changes at a postsynaptic locus as a result of associative pre- and postsynaptic activity.

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

Two types of calcium response limited to single spines in cerebellar Purkinje cells.

Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.

A methodology for improving computed individual regressions predictions.

This research proposes a methodology to improve computed individual prediction values provided by an existing regression model without having to change either its parameters or its architecture. In other words, we are interested in achieving more accurate results by adjusting the calculated regression prediction values, without modifying or rebuilding the original regression model. Our proposition is to adjust the regression prediction values using individual reliability estimates that indicate if a single regression prediction is likely to produce an error considered critical by the user of the regression. The proposed method was tested in three sets of experiments using three different types of data. The first set of experiments worked with synthetically produced data, the second with cross sectional data from the public data source UCI Machine Learning Repository and the third with time series data from ISO-NE (Independent System Operator in New England). The experiments with synthetic data were performed to verify how the method behaves in controlled situations. In this case, the outcomes of the experiments produced superior results with respect to predictions improvement for artificially produced cleaner datasets with progressive worsening with the addition of increased random elements. The experiments with real data extracted from UCI and ISO-NE were done to investigate the applicability of the methodology in the real world. The proposed method was able to improve regression prediction values by about 95% of the experiments with real data.

Optical and ultraviolet observations of a strong flare in the young, single K2 dwarf LQ Hya

We present high-resolution optical echelle spectra and IUE observations during a strong flare on 1993 December 22 in the very active, young, rapidly rotating, single K2 dwarf LQ Hya. The initial impulsive phase of the flare, which started sometime between 2:42 ut and 4:07 ut, was characterized by strong optical continuum enhancement and blueshifted emission lines with broad wings. The optical chromospheric lines reached their maximum intensity at ≈ 5:31 ut, by which time the blueshift vanished and the optical continuum enhancement had sharply decreased. Thereafter, the line emission slowly decreased and the lines redshift in a gradual phase that lasted at least two more hours. The Mg II lines behaved similarly. Quiescent C IV flux levels were not recovered until 21 h later, though a data gap and a possible second flare make the interpretation uncertain. In addition to the typically flare-enhanced emission lines (e.g., H α and H β), we observe He I D_3 going into emission, plus excess emission (after subtraction of the quiescent spectrum) in other He I and several strong neutral metal lines (e.g., Mg I b). Flare enhancement of the far-ultraviolet continuum generally agrees with an Si I recombination model. We estimate the total flare energy, and discuss the broad components, asymmetries and Doppler shifts seen in some of the emission lines.

Deep observation of the NGC1275 region with MAGIC: search of diffuse gamma-ray emission from cosmic rays in the Perseus cluster

Clusters of galaxies are expected to be reservoirs of cosmic rays (CRs) that should produce diffuse γ-ray emission due to their hadronic interactions with the intra-cluster medium. The nearby Perseus cool-core cluster, identified as the most promising target to search for such an emission, has been observed with the MAGIC telescopes at very-high energies (VHE, E ≥ 100 GeV) for a total of 253 hr from 2009 to 2014. The active nuclei of NGC 1275, the central dominant galaxy of the cluster, and IC 310, lying at about 0.6º from the centre, have been detected as point-like VHE γ-ray emitters during the first phase of this campaign. We report an updated measurement of the NGC 1275 spectrum, which is described well by a power law with a photon index Γ = 3.6 ± 0.2_(stat) ± 0.2_(syst) between 90 GeV and 1200 GeV. We do not detect any diffuse γ-ray emission from the cluster and so set stringent constraints on its CR population. To bracket the uncertainties over the CR spatial and spectral distributions, we adopt different spatial templates and power-law spectral indexes α. For α = 2.2, the CR-to-thermal pressure within the cluster virial radius is constrained to be ≤ 1 − 2%, except if CRs can propagate out of the cluster core, generating a flatter radial distribution and releasing the CR-to-thermal pressure constraint to ≤ 20%. Assuming that the observed radio mini-halo of Perseus is generated by secondary electrons from CR hadronic interactions, we can derive lower limits on the central magnetic field, B_(0), that depend on the CR distribution. For α = 2.2, B_(0) ≥ 5 − 8 µG, which is below the ∼25 µG inferred from Faraday rotation measurements, whereas for α ≤ 2.1, the hadronic interpretation of the diffuse radio emission contrasts with our γ-ray flux upper limits independently of the magnetic field strength.

Optical probing of spin fluctuations of a single paramagnetic Mn atom in a semiconductor quantum dot

We analyzed the photoluminescence intermittency generated by a single paramagnetic spin localized in an individual semiconductor quantum dot. The statistics of the photons emitted by the quantum dot reflect the quantum fluctuations of the localized spin interacting with the injected carriers. Photon correlation measurements, which are reported here, reveal unique signatures of these fluctuations. A phenomenological model is proposed to quantitatively describe these observations, allowing a measurement of the spin dynamics of an individual magnetic atom at zero magnetic field. These results demonstrate the existence of an efficient spin-relaxation channel arising from a spin exchange with individual carriers surrounding the quantum dot. A theoretical description of a spin-flip mechanism involving spin exchange with surrounding carriers gives relaxation times in good agreement with the measured dynamics.

Hot thermal X-ray emission from the Be star HD 119682

We present an analysis of a series of four consecutive Chandra high-resolution transmission gratings observations, amounting to a total of 150 ks, of the Be X-ray source HD 119682 (=1WGA J1346.5–6255), a member of the new class of γ Cas analogs. The Chandra light curve shows significant brightness variations on timescales of hours. However, the spectral distribution appears rather stable within each observation and during the whole campaign. A detailed analysis is not able to detect any coherent pulsation up to a frequency of 0.05 Hz. The Chandra High Energy Transmission Gratings spectrum seems to be devoid of any strong emission line, including Fe Kα fluorescence. The continuum is well described with the addition of two collisionally ionized plasmas of temperatures kT ≈ 15 keV and 0.2 keV, respectively, by the apec model. Models using photoionized plasma components (mekal) or non-thermal components (powerlaw) give poorer fits, providing support for the pure thermal scenario. These two components are absorbed by a single column with N H = (0.20+0.15 –0.03) × 1022 cm–2 compatible with the interstellar value. We conclude that HD 119682 can be regarded as a pole-on γ Cas analog.

Modelling of the surface emission of the low magnetic field magnetar SGR 0418+5729

We perform a detailed modelling of the post-outburst surface emission of the low magnetic field magnetar SGR 0418+5729. The dipolar magnetic field of this source, B=6×1012G estimated from its spin-down rate, is in the observed range of magnetic fields for normal pulsars. The source is further characterized by a high pulse fraction and a single-peak profile. Using synthetic temperature distribution profiles, and fully accounting for the general-relativistic effects of light deflection and gravitational redshift, we generate synthetic X-ray spectra and pulse profiles that we fit to the observations. We find that asymmetric and symmetric surface temperature distributions can reproduce equally well the observed pulse profiles and spectra of SGR 0418. None the less, the modelling allows us to place constraints on the system geometry (i.e. the angles ψ and ξ that the rotation axis makes with the line of sight and the dipolar axis, respectively), as well as on the spot size and temperature contrast on the neutron star surface. After performing an analysis iterating between the pulse profile and spectra, as done in similar previous works, we further employed, for the first time in this context, a Markov-Chain Monte Carlo approach to extract constraints on the model parameters from the pulse profiles and spectra, simultaneously. We find that, to reproduce the observed spectrum and flux modulation: (a) the angles must be restricted to 65° ≲ ψ + ξ ≲ 125° or 235° ≲ ψ + ξ ≲ 295°; (b) the temperature contrast between the poles and the equator must be at least a factor of ∼6, and (c) the size of the hottest region ranges between 0.2 and 0.7 km (including uncertainties on the source distance). Lastly, we interpret our findings within the context of internal and external heating models.

Single planktonic foraminiferal Mg/Ca ratios of samples from sediment trap CBi3 upper off Cape Blanc (NW-Africa)

The data shows Mg/Ca ratios of single specimens of the planktonic foraminifera Globigerinoides ruber (pink and white variety) and Globorotalia Inflata from a sediment trap anchored off Cape Blanc (NW-Africa). Mg/Ca ratios were determined on an ICP-MS and ICP-OES at the University of Bremen, after flow-through cleaning. Additionally, shells sizes and calculated shell weights are provided.

Human brain regions required for the dividing and switching of attention between two features of a single object

This combined PET and ERP study was designed to identify the brain regions activated in switching and divided attention between different features of a single object using matched sensory stimuli and motor response. The ERP data have previously been reported in this journal [64]. We now present the corresponding PET data. We identified partially overlapping neural networks with paradigms requiring the switching or dividing of attention between the elements of complex visual stimuli. Regions of activation were found in the prefrontal and temporal cortices and cerebellum. Each task resulted in different prefrontal cortical regions of activation lending support to the functional subspecialisation of the prefrontal and temporal cortices being based on the cognitive operations required rather than the stimuli themselves. (C) 2003 Elsevier Science B.V. All rights reserved.

Stationary two-atom entanglement induced by nonclassical two-photon correlations

A system of two two-level atoms interacting with a squeezed vacuum field can exhibit stationary entanglement associated with nonclassical two-photon correlations characteristic of the squeezed vacuum field. The amount of entanglement present in the system is quantified by the well known measure of entanglement called concurrence. We find analytical formulae describing the concurrence for two identical and nonidentical atoms and show that it is possible to obtain a large degree of steady-state entanglement in the system. Necessary conditions for the entanglement are nonclassical two-photon correlations and nonzero collective decay. It is shown that nonidentical atoms are a better source of stationary entanglement than identical atoms. We discuss the optimal physical conditions for creating entanglement in the system; in particular, it is shown that there is an optimal and rather small value of the mean photon number required for creating entanglement.