A persistent ultraviolet outflow from an accreting neutron star binary transient.

All disc-accreting astrophysical objects produce powerful disc winds. In compact binaries containing neutron stars or black holes, accretion often takes place during violent outbursts. The main disc wind signatures during these eruptions are blue-shifted X-ray absorption lines, which are preferentially seen in disc-dominated 'soft states'1,2. By contrast, optical wind-formed lines have recently been detected in 'hard states', when a hot corona dominates the luminosity3. The relationship between these signatures is unknown, and no erupting system has as yet revealed wind-formed lines between the X-ray and optical bands, despite the many strong resonance transitions in this ultraviolet (UV) region4. Here we report that the transient neutron star binary Swift J1858.6-0814 exhibits wind-formed, blue-shifted absorption lines associated with C IV, N V and He II in time-resolved UV spectroscopy during a luminous hard state, which we interpret as a warm, moderately ionized outflow component in this state. Simultaneously observed optical lines also display transient blue-shifted absorption. Decomposing the UV data into constant and variable components, the blue-shifted absorption is associated with the former. This implies that the outflow is not associated with the luminous flares in the data. The joint presence of UV and optical wind features reveals a multi-phase and/or spatially stratified evaporative outflow from the outer disc5. This type of persistent mass loss across all accretion states has been predicted by radiation-hydrodynamic simulations6 and helps to explain the shorter-than-expected duration of outbursts7.

Recent advances in nonlinear microscopy: Deep insights and polarized revelations.

Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.

Parental review of service uptake during the pre-school period.

This article outlines the development and analysis of the results of a community-led survey undertaken in year 1 of a 3-year period of evaluation for a first wave Sure Start programme. The survey had initially been suggested as a means of collecting data that was required, at the time, by the central Sure Start unit, however, its potential outside of this specific evaluative purpose was also recognized. In addition to providing important pointers for the future development of the programme, the findings give insight into a community within the west midland conurbation, and reviews the support that carers have accessed during the first 4 years of their youngest child's life. Their feelings and opinions are presented and demonstrate factors that influence the involvement of carers in this and other related programmes.

Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis.

Growth of new micrometre sized projections called dendritic spines in neurones has been linked to the encoding of long-term memories in vertebrates. Numerous studies have been carried out at both the light and electron microscopy level to quantify dendritic spine densities in brain tissue in laboratory animals. Currently, such efforts using light microscopy have relied on manual counting of spines in confocal or two-photon optical slice images of tissue containing fluorescently labelled spines. This manual approach can be slow and tedious, especially for samples with high spine densities. We introduce an alternative way of performing spine counting that uses an applied image intensity threshold followed by spatial image correlation spectroscopy (ICS) analysis. We investigated the effect of particle sizes above the diffraction limit on the autocorrelation analysis as well as the influence of background fluorescence. Our results show that, for well labelled cerebellar tissue samples imaged with a signal-to-noise ratio of 5 or greater, ICS-based spine counts can be conducted with the same 15-20% precision as manual counting, but much more rapidly.

Two-photon image correlation spectroscopy and image cross-correlation spectroscopy.

We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coefficients as large as 10(-8) cm2 s(-1) that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy. We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.

Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy.

We demonstrate a widefield multiphoton microscope and a temporally decorrelated, multifocal, multiphoton microscope that is based on a high-efficiency array of cascaded beamsplitters. Because these microscopes use ultrashort pulse excitation over large areas of the sample, they allow efficient use of the high-average power available from modern ultrashort pulse lasers.

Image correlation spectroscopy. II. Optimization for ultrasensitive detection of preexisting platelet-derived growth factor-beta receptor oligomers on intact cells.

Previously we introduced image correlation spectroscopy (ICS) as an imaging analog of fluorescence correlation spectroscopy (FCS). Implementation of ICS with image collection via a standard fluorescence confocal microscope and computer-based autocorrelation analysis was shown to facilitate measurements of absolute number densities and determination of changes in aggregation state for fluorescently labeled macromolecules. In the present work we illustrate how to use ICS to quantify the aggregation state of immunolabeled plasma membrane receptors in an intact cellular milieu, taking into account background fluorescence. We introduce methods that enable us to completely remove white noise contributions from autocorrelation measurements for individual images and illustrate how to perform background corrections for autofluorescence and nonspecific fluorescence on cell population means obtained via ICS. The utilization of photon counting confocal imaging with ICS analysis in combination with the background correction techniques outlined enabled us to achieve very low detection limits with standard immunolabeling methods on normal, nontransformed human fibroblasts (AG1523) expressing relatively low numbers of platelet-derived growth factor-beta (PDGF-beta) receptors. Specifically, we determined that the PDGF-beta receptors were preaggregated as tetramers on average with a mean surface density of 2.3 clusters micrometer(-2) after immunolabeling at 4 degreesC. These measurements, which show preclustering of PDGF-beta receptors on the surface of normal human fibroblasts, contradict a fundamental assumption of the ligand-induced dimerization model for signal transduction and provide support for an alternative model that posits signal transduction from within preexisting receptor aggregates.

Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source.

We demonstrate the first use, to our knowledge, of a compact, diode-pumped, femtosecond fiber laser for third-harmonic generation (THG) microscopy. We discuss the utility of this technique, as well as the technical issues involved in using this compact source, and demonstrate the first use, to our knowledge, of imaging by THG backlighting.

Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy.

Communication between cells invariably involves interactions of a signalling molecule with a receptor at the surface of the cell. Typically, the receptor is imbedded in the membrane and it is hypothesized that the binding of the signalling molecule causes a change in the state of aggregation of the receptor which, in turn, initiates a biochemical signal within the cell. Subsequently, many of the occupied receptors bind to membrane-associated structures, called coated pits, which invaginate and pinch off to form coated vesicles, thereby removing the receptors from the cell surface. The state of aggregation of membrane receptors is obviously in constant flux. Any useful approach to measuring the state of aggregation must, therefore, allow for dynamic measurements in living cells. It is possible to use fluorescently labelled signalling molecules or antibodies directed at the receptor of interest to visualize the receptor on the cell surface with a fluorescence microscope. By employing a laser confocal microscope, high resolution images can be produced in which the fluorescence intensity is quantitatively imaged as a function of position across the surface of the cell. Calculations of autocorrelation functions of these images provide direct and accurate measures of the density of fluorescent particles on the surface. Combined with the average intensity in the image, which reflects the total average number of molecules, it is possible to estimate the degree of aggregation of the receptor molecules. We refer to this analysis as image correlation spectroscopy (ICS). We show how ICS can be used to measure the density of several receptors on a variety of cells and how it can be used to measure the density of coated pits and the number of molecules per coated pit. We also show how the technique can be used to monitor fusion of virus particles to cell membranes. Further, we illustrate that, by calculating cross-correlation functions between pairs of images, we can extend the analysis to measurements of the distributions as a function of time, on the second timescale, as well as to measurements of the movement of the receptor aggregates on the surface. Finally, we illustrate that, by this approach, we can measure the extent of interaction between two different receptors as a function of time. This represents the most quantitative measurement of the extent of co-localization of receptors available and is independent of the spatial resolution of the confocal microscope. The theory of ICS and image cross-correlation spectroscopy (ICCS), focussing on the interpretation of the data in terms of the biological phenomenon being probed, is discussed.

Aggregation of PDGF-beta receptors in human skin fibroblasts: characterization by image correlation spectroscopy (ICS).

Receptor aggregation is believed to be an important, early step when growth factors such as PDGF stimulate proliferation and differentiation of cell populations. To investigate receptor aggregation, we utilized a novel biophysical technique, image correlation spectroscopy, to study the distribution and aggregation state of PDGF-beta receptors on the surface of human dermal fibroblasts under various experimental conditions. It was found that the cell surface receptors were pre-clustered at 4 degrees C and receptor aggregation increased for samples measured at 37 degrees C. Treatment with PDGF-BB had no measurable effect on the receptor aggregation state. The results also indicate that additions of 10% serum or an inhibitor of tyrosine kinase activity, may disperse the receptors. The results of this study are consistent with organization of PDGF-beta receptors in pre-existing membrane domains.

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application.

Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.

Terodiline with bladder retraining for treating detrusor instability in elderly people.

OBJECTIVE: To compare terodiline with bladder retraining against placebo with bladder retraining in the treatment of detrusor instability in frail elderly patients. DESIGN: Randomised, double blind, parallel group study. Treatment lasted for six weeks. Frequency of micturition and episodes of incontinence recorded on diary chart by patients. SETTING: Incontinence clinic and a geriatric day hospital at two teaching hospitals. PATIENTS: 37 frail but ambulant patients, mean (range) age 80.4 (70-89) years with urinary frequency and urge incontinence, due to detrusor instability. Two patients withdrew before the first assessment (one in each group) and one could not complete the diary chart (placebo group). INTERVENTIONS: 19 patients received bladder retraining and terodiline 25 mg daily and 18 bladder retraining and placebo. MAIN OUTCOME MEASURES: Change in urinary frequency and number of episodes of incontinence after six weeks' treatment. Patient's subjective evaluation of symptoms. RESULTS: Little difference was found in the results of treatment with terodiline and placebo. The change in episodes of incontinence per 24 hours was no different in the two groups (95% confidence interval -0.6 to 1.2; p = 0.75) and the difference between treatments in the change in frequency of micturition per 24 hours (-0.2) was not significant (-1.1 to 1.2; p = 0.76). Ten patients taking terodiline thought they had improved compared with seven receiving placebo; this difference was not significant. CONCLUSION: Although the number of patients in each group was small and may have been insufficient to detect a drug effect, the possible benefit of terodiline is likely to be small.

The demonstration of saphenofemoral incompetence; doppler ultrasound compared with standard clinical tests.

Patients with primary varicose veins were examined by a combination of the standard tourniquet test with detection of reflux by Doppler ultrasound. Results were compared with standard clinical tests: impulse or thrill at the saphenous opening on coughing, tap impulse at the groin, and the 'Trendelenburg' tourniquet test. The state of competence of the saphenofemoral junction was noted at operation. One hundred and sixty-one limbs of 105 patients were studied. The saphenofemoral junction was incompetent in 132/161 limbs (82 per cent) and was judged competent in 29/161 limbs (18 per cent). The combined Doppler and tourniquet test assessed the saphenofemoral junction correctly in 82 per cent of limbs and was more accurate than all the other tests. The test had good sensitivity (0.9) but poor specificity (0.45). Poor specificity was a feature of all the tests except for thrill which was a highly insensitive test. The combined Doppler and tourniquet test appears to be the most simple, rapid and accurate means of detecting saphenofemoral incompetence.