Ms Time Resolution Research Articles

In Situ Au L3 and L2 edge XANES spectral analysis during growth of thiol protected gold nanoparticles for the study on particle size dependent electronic properties

The formation of gold nanoparticles in the presence of dodecanethiol was observed in situ by quick X-ray absorption fine structure spectroscopy with 100 ms time resolution at the Au L 3 and L 2 edges. The series of the X-ray absorption near edge structure spectra at both edges was analyzed to evaluate the dispersion and electronic properties of gold nanoparticles during their growth.

Sub-Millisecond Time-Resolved 2-Dimensional X-Ray Diffraction Recording from Stretch-Activated Flight Muscle from Bumblebee

We have previously recorded the responses of X-ray reflections from bumblebee flight muscle (IFM) fibers to step length changes at a 3.4 ms time resolution (Iwamoto et al., Biophys. J., 2010). This study showed that, upon stretch, the myosin heads supporting isometric force detached, and were dynamically replaced by new myosin heads recruited by stretch activation (SA). At the same time, tropomyosin molecules (TM) temporarily moved back to its inactivating positions. These motions of proteins were very fast, and clearly the time resolution of the 3-CCD detector (640 x 480 pixels, 3.4 ms/full-frame) was insufficient. Here we used a new high-speed CMOS detector (1024 x 1024 pixels, up to 0.2 ms/full-frame) and repeated time-resolved measurements with a 0.5-ms time resolution. Unlike CCD detectors, the CMOS detector does not have to transmit charges serially, enabling a fast readout. In addition, we have improved the experimental design so that the IFM fibers tolerate higher X-ray dose (thus higher signal). Mitochondrial remnants, a major diffusion barrier for substances, were thoroughly removed to ensure sufficient ATP supply. After these improvements, the analysis from fully Ca-activated fibers showed that the stretch-induced detachment of myosin heads supporting isometric force is complete within 1 ms after completion of a 1-ms stretch, as shown by the reciprocal intensity changes of troponin reflections. The reattachment of myosin heads was slightly faster than the rise of SA force, and the number of attached heads started to decrease while the force was still rising. The movement of tropomyosin was perfectly synchronized with myosin detachment/reattachment, making it likely that tropomyosin and wings are beating synchronously in insects with “asynchronous” IFM. However, signals that trigger SA were not detected in the tropomyosin reflection even with this time resolution.

Fast Topology Changes During SNARE-Mediated Vesicle Fusion Observed in Supported Membranes by Polarized Tirfm

In vitro reconstitution experiments have played an essential role in a large body of research on SNARE-mediated membrane fusion. Recently, new single vesicle assays have been developed to gain more detailed insight into the kinetics of vesicle docking and fusion. Previously, we used supported membranes in combination with total internal reflection microscopy (TIRFM) to record the docking and fusion of synaptobrevin containing vesicles with 4 ms time resolution. Depending on the lipid conditions, we found that between 3 and 8 SNARE complexes are needed for fast fusion in this system. Here we utilize polarized TIRFM to investigate topology changes that the docked vesicles undergo after the onset of fusion. The theory that describes the fluorescence intensity during the transformation of a single vesicle from a spherical particle to a flat membrane patch is developed and confirmed by experiments with the three fluorescent probes Rh-DOPE, NBD-DPPE and BODIPY-PC. Our results show that, on average, the fusing vesicles flatten and merge into the planar membrane within 8 ms after fusion starts.

High-Throughput Screening of Response Kinetics of Genetically-Encoded Metal Sensors with Microfluidics Technology

Metal sensors, chromophores which generate responses upon metal-ion binding, are indispensible tools to study the metal homeostasis of the cell. Compared to the traditional dye-based metal sensors, the fluorescent protein (FP)-based FRET sensors offer many distinctive advantages. They are fully genetically encoded and targeted to specific cell location, capable of controlled expression, and provide intensity-independent ratiometric measurement. Yet at present the performances of the FP-based sensors, such as the dynamic range, metal selectivity, and metal affinity, need to be markedly improved to meet the requirements and challenges of their cellular application. By combining a sensor library design strategy, the aim of our work is to screen and sort the FP-based sensors for improved photophysical and photochemical properties using a high-throughput microfluidics method. We have developed a microfluidic platform that combines high-throughput screening with versatile optical FRET detection, fast mixing, and laser trapping techniques on individual mammalian cells. We demonstrated that the FRET responses of the sensors can readily be measured and differentiated on our cytometer with a rate of >20 cells/second. At this throughput a typical small targeted sensor library (∼105 clones) can be sorted within a time frame of several hours. Using this technique we also investigated the response kinetics of various Ca2+ and Zn2+ sensors expressed at different cell locations upon exposure to metal ions in the microfluidic environment. Dissection of the relationship between sensor kinetics, construction, and location will shed light on the mechanism and dynamics of sensor function and ion transportation in the cell. The high-throughput characteristics of the microfluidics approach, integrated with fast optical detection with ms time-resolution over a broad time window (spanning orders of magnitudes from ms to seconds), can be readily adapted to other high-fidelity kinetics-based screening or sorting applications.

Single Molecule FRET Studies on Kinetics of Elongation Factor Tu Binding to the Ribosome during the tRNA Selection Process

Elongation factor Tu (EF-Tu) ensures fidelity in protein synthesis by mediating the entry of cognate aminoacyl-tRNA (aa-tRNA) into the A-site of the ribosome via formation of an EF-Tu.GTP.aa-tRNA ternary complex (TC). In order to probe the kinetic details of EF-Tu interactions with both aminoacyl tRNA and the ribosome during the tRNA selection process, we have constructed, purified, and labeled an E. coli EF-Tu mutant at position 348 (E348C) with either a fluorescence quencher (QSY9) or a fluorescent dye (Cy3 or Cy5). This position of labeling allows monitoring of EF-Tu interactions with fluorescent derivatives of ribosomal protein L11 (labeled at position 87) and aa-tRNA (labeled in the dihydroU loop). These three positions form an almost equilateral triangle within the ribosome, at distances that are appropriate for sensitive monitoring by single molecule fluorescence resonance energy transfer (smFRET). Two kinetic steps, denoted as TC association (4.7±0.3×107 M−1s−1) and EF-Tu dissociation (12±1 s−1), are found with Cy3-Cy5 FRET pairs or Cy3-QSY9 pairs placed on L11/EF-Tu, tRNA/EF-Tu (A-site) or tRNA/tRNA (A-site/P-site) pairs. The reaction rates are almost independent of labeling strategy and agree with ensemble measurements. At 10 ms time resolution, the FRET between L11/EF-Tu and tRNA/EF-Tu (A-site) pairs showed only one EF-Tu bound conformational state that is detectable during the tRNA selection process, providing strong evidence that, at this time resolution, EF-Tu loses proximity essentially simultaneously with both L11 and aa-tRNA. Alternating-laser excitation experiments demonstrate that, following EF-Tu dissociation, a fraction of aa-tRNA remains stably bound to the ribosome, corresponding to aa-tRNA that has successfully accommodated into the A-site, while the remainder dissociates rapidly, presumably due to rejection via proofreading.Supported by NIH (GM080376, GM071014 and HG004364) and NIST (70NANB7H0711).

Measure of electron cyclotron emission at multiple angles in high Te plasmas of JET

The oblique electron cyclotron emission (ECE) diagnostic installed at JET allows simultaneous analysis of the ECE spectra along three lines of sight (with toroidal angles of 0°, ∼ 10°, and ∼ 20°) and two linear polarizations for each oblique line of sight. The diagnostic is capable of measuring EC emission over the band of 75–800 GHz with 5 ms time resolution and 7.5 GHz spectral resolution, and it is designed to investigate the features of ECE spectra related to electron distribution in the thermal velocity range. Instrumental accuracy was assessed using sources at different temperatures (77–900 K) and with plasma emission. ECE from high temperature plasmas and in the presence of fast ions has been compared to simulations performed with the modeling code SPECE, setting an upper limit to possible discrepancies from thermal emission.

Recent developments of the JET far-infrared interferometer-polarimeter diagnostic

The far-infrared diagnostic provides essential internal measurements of the plasma density and magnetic field topology (q-profile via Faraday rotation angle) in real-time. The diagnostic capabilities have recently been extended in a number of key areas. Fast interferometer data, with 10 μs time resolution, and a new MATLAB code have allowed improved analysis of the evolution of density profiles during fast events such as vertical plasma displacements, edge localized mode, pellet fuelling, and disruptions. Using the polarimeter measurements in real-time, a new calibration procedure has been developed based on a propagation code using the Mueller matrix formalism. A further major upgrade of the system is presently underway: adding a second color laser to the vertical channels and implementing a new phase counter based on analog zero crossing and field-programmable gate array boards.

Fast neutron-gamma discrimination on neutron emission profile measurement on JT-60U

A digital signal processing (DSP) system is applied to stilbene scintillation detectors of the multichannel neutron emission profile monitor in JT-60U. Automatic analysis of the neutron-γ pulse shape discrimination is a key issue to diminish the processing time in the DSP system, and it has been applied using the two-dimensional (2D) map. Linear discriminant function is used to determine the dividing line between neutron events and γ-ray events on a 2D map. In order to verify the validity of the dividing line determination, the pulse shape discrimination quality is evaluated. As a result, the γ-ray contamination in most of the beam heating phase was negligible compared with the statistical error with 10 ms time resolution.

Detailed analysis of latencies in image‐based dynamic MLC trackinga)

Previous measurements of the accuracy of image-based real-time dynamic multileaf collimator (DMLC) tracking show that the major contributor to errors is latency, i.e., the delay between target motion and MLC response. Therefore the purpose of this work was to develop a method for detailed analysis of latency contributions during image-based DMLC tracking. A prototype DMLC tracking system integrated with a linear accelerator was used for tracking a phantom with an embedded fiducial marker during treatment delivery. The phantom performed a sinusoidal motion. Real-time target localization was based on x-ray images acquired either with a portal imager or a kV imager mounted orthogonal to the treatment beam. Each image was stored in a file on the imaging workstation. A marker segmentation program opened the image file, determined the marker position in the image, and transferred it to the DMLC tracking program. This program estimated the three-dimensional target position by a single-imager method and adjusted the MLC aperture to the target position. Imaging intervals deltaT(image) from 150 to 1000 ms were investigated for both kV and MV imaging. After the experiments, the recorded images were synchronized with MLC log files generated by the MLC controller and tracking log files generated by the tracking program. This synchronization allowed temporal analysis of the information flow for each individual image from acquisition to completed MLC adjustment. The synchronization also allowed investigation of the MLC adjustment dynamics on a considerably finer time scale than the 50 ms time resolution of the MLC log files. For deltaT(image) = 150 ms, the total time from image acquisition to completed MLC adjustment was 380 +/- 9 ms for MV and 420 +/- 12 ms for kV images. The main part of this time was from image acquisition to completed image file writing (272 ms for MV and 309 ms for kV). Image file opening (38 ms), marker segmentation (4 ms), MLC position calculation (16 ms), and MLC adjustment (52 ms) were considerably faster. For deltaT(image) = 1000 ms, the total time from image acquisition to completed MLC adjustment increased to 1030 +/- 62 ms (MV) and 1330 +/- 52 ms (kV) mainly because of delayed image file writing. The MLC adjustment duration was constant 52 ms (+/- 3 ms) for MLC adjustments below 1.1 mm and increased linearly for larger MLC adjustments. A method for detailed time analysis of each individual real-time position signal for DMLC tracking has been developed and applied to image-based tracking. The method allows identification of the major contributors to latency and therefore a focus for reducing this latency. The method could be an important tool for the reconstruction of the delivered target dose during DMLC tracking as it provides synchronization between target motion and MLC motion.

Mesodynamics: watching vesicle formation in situ by small-angle neutron scattering

Self-aggregating systems form a large variety of different structures depending on type and concentration of the amphiphiles. Morphological changes can be triggered by mixing two surfactant solutions with the stopped-flow technique and observing by highly time-resolved SANS experiments, thereby allowing to obtain of structural information with 25–50 ms time resolution. Here, the formation of unilamellar vesicles by mixing zwitterionic tetradecyldimethylamine oxide (TDMAO) with the anionic lithium perfluorooctanesulfonate (LiPFOS) was studied as a function of the mixing ratio. SANS data were analyzed in terms of the structural changes and especially with respect to intermediate structures. Vesicle formation occurs via disk-like micelles as intermediates that close to form very monodisperse unilamellar vesicles after reaching a certain size. This process is controlled by the competition between bending energy of the bilayer and the line energy of the disk rim, and its characteristic time is controlled by the electrostatic repulsion that is determined by the TDMAO/LiPFOS ratio.

Mapping of Fluidic Mixing in Microdroplets with 1 μs Time Resolution Using Fluorescence Lifetime Imaging

Microdroplets generated in microfluidic channels hold great promise for use as substrates in high-throughput chemical and biological analysis. These water-in-oil compartments can serve as isolated reaction vessels, and since they can be generated at rates in excess of 1 kHz, thousands of assays can be carried out quickly and reproducibly. Nevertheless, sampling the large amount of information generated from these platforms still remains a significant challenge. For example, considering the high droplet generation rates and velocities, reproducibility and micrometer resolution are challenging requirements that must be fulfilled. Herein we combine confocal fluorescence lifetime imaging microscopy with a statistical implementation that permits the analysis of mixing phenomena within microdroplets with a temporal resolution of 1 mus. Importantly, such exquisite resolution is only possible as a result of the large number of droplets sampled and their high structural reproducibility.

Oxide mediated liquid–solid growth of high aspect ratio aligned gold silicide nanowires onSi(110) substrates

Silicon nanowires grown using the vapor–liquid–solid method are promising candidates fornanoelectronics applications. The nanowires grow from an Au–Si catalyst during siliconchemical vapor deposition. In this paper, the effect of temperature, oxide at the interfaceand substrate orientation on the nucleation and growth kinetics during formation ofnanogold silicide structures is explained using an oxide mediated liquid–solidgrowth mechanism. Using real time in situ high temperature transmission electronmicroscopy (with 40 ms time resolution), we show the formation of high aspect ratio (≈15.0) aligned gold silicide nanorods in the presence of native oxide at the interface during in situannealing of gold thin films on Si(110) substrates. Steps observed in the growth rate andreal time electron diffraction show the existence of liquid Au–Si nano-alloy structures onthe surface besides the un-reacted gold nanostructures. These results might enable us toengineer the growth of nanowires and similar structures with an Au–Si alloy as a catalyst.

Compact High-Speed Spectrometers Based on MEMS Devices with Large Amplitude In-Plane Actuators

At present most spectrometers, in particular for wavelengths longer than the VIS, are comparatively large, stationary and expensive. With the advent of micro-electro-mechanical systems (MEMS), it became possible to build pocket-sized spectrometers for various spectral ranges, including the near-IR or mid-IR. These systems are highly rugged and can measure spectral changes at ms time resolution or to co-add several hundreds of scans to one spectrum achieving adequate signal-to-noise ratios. Two spectrometer systems a Czerny-Turner type scanning monochromator and a FT-IR spectrometer both based on a micromechanical scanning mirror technology with in plane electrostatic actuation are presented.

Heat loads on plasma facing components during disruptions on JET

For the first time, fast measurements of heat loads on the main chamber plasma facing components (about 1 ms time resolution) during disruptions are taken on JET. The timescale of energy deposition during the thermal quench is estimated and compared with the timescale of the core plasma collapse measured with soft x-ray diagnostic. The energy deposition time is 3–8 times longer than the plasma energy collapse during density limit disruptions or radiative limit disruptions. This factor is rather in the range 1.5–4 for vertical displacement events. The heat load profiles measured during the thermal quench show substantial broadening of the power footprint on the upper dump plate. The scrape-off layer power width is increased by a factor of 3 for the density limit disruptions. The far scrape-off layer is characterized by a steeper gradient which could be explained by shadowing of the dump plate by other main chamber plasma facing components such as the outer limiter.

Applying Dynamic and Synchronous DRIFTS/EXAFS to the Structural Reactive Behaviour of Dilute (≤1 wt%) Supported Rh/Al2O3 Catalysts using Quick and Energy Dispersive EXAFS

Examples of the application of a synchronous DRIFTS/EXAFS/mass spectrometry (MS) methodology to the study of dilute (≤ 1wt%) Rh/Al2O3 catalysts are discussed. These are used to explore the potential of this approach for understanding of the behaviour of supported metal catalysts “in a single shot”, and in the often preferred regime of low (<1 wt%) loadings of active precious metals. Firstly, the sequential interaction of NO (323 K) and then CO (373 K) with reduced, 0.5 wt% Rh/Al2O3 catalysts is studied. Infrared spectroscopy indicates that two surface species (a bent Rh(NO−) and Rh(CO)2 species) can be created using this sequential gas absorption/reaction method with minimal interference from other carbonyl or nitrosyl species. As such the potential for a reliable structural characterisation of the local structure of these species by EXAFS becomes possible. However, in contrast to the infrared spectroscopy, analysis of the EXAFS data also indicates that, even for such low loaded Rh systems, oxidative disruption of the Rh by the NO and CO is not complete and that bonding typical of small Rh clusters persists in both cases. The possible sources of this apparent spectroscopic difference of opinion are discussed. Secondly, 1wt% Rh/Al2O3 catalysts are studied using dispersive EXAFS at 573 K with 100 ms time resolution, during a redox switching event involving a reducing feedstock comprising just 3000 ppm of CO and 3000 ppm of NO. It is shown that highly useful and insightful time resolved and synchronously obtained XANES/EXAFS/IR data can be obtained even in this dilute Rh and more “realistic” case. Additional data, regarding the overall performance of the experiment, as currently implemented at the ESRF, along with a discussion of where enhanced performance might be yet still be gained, are also given.

The Mini-Calorimeter on-board AGILE: The first year in space

AGILE, the Italian space mission dedicated to gamma-ray and hard-X astrophysics, was successfully launched on 23rd April 2007 and is currently fully operative. The Mini-Calorimeter (MCAL) on-board the AGILE satellite is a scintillation detector made of 20 kg of segmented CsI(Tl) scintillator with photodiode readout with a total geometrical area of 1400 cm2. MCAL can work both as a slave of the AGILE Silicon tracker and as an independent detector for gamma-ray bursts (GRB) detection in the 300 keV - 100 MeV energy range. Despite its limited thickness, due to weight constraints, MCAL has proven to successfully self-trigger GRBs at MeV energies providing photon-by-photon data with less than 2 μs time resolution and almost all-sky detection capabilities. The instrument design and characteristics, as well as the in-flight performance after one year of operation in space and the scientific results obtained so far are reviewed and discussed.

Spectroscopic measurements of temperature and plasma impurity concentration during magnetic reconnection at the Swarthmore Spheromak Experiment

Electron temperature measurements during counterhelicity spheromak merging studies at the Swarthmore Spheromak Experiment (SSX) [M. R. Brown, Phys. Plasmas 6, 1717 (1999)] are presented. VUV monochromator measurements of impurity emission lines are compared with model spectra produced by the non-LTE excitation kinematics code PRISMSPECT [J. J. MacFarlane et al., in Proceedings of the Third Conference on Inertial Fusion Science and Applications (2004)] to yield the electron temperature in the plasma with 1 μs time resolution. Average Te is seen to increase from 12 to 19 eV during spheromak merging. Average C III ion temperature, measured with a new ion Doppler spectrometer (IDS) [C. D. Cothran et al., Rev. Sci. Instrum. 77, 063504 (2006)], likewise rises during spheromak merging, peaking at ∼22 eV, but a similar increase in Ti is seen during single spheromak discharges with no merging. The VUV emission line measurements are also used to constrain the concentrations of various impurities in the SSX plasma, which are dominated by carbon, but include some oxygen and nitrogen. A burst of soft x-ray emission is seen during reconnection with a new four-channel detector (SXR). There is evidence for spectral changes in the soft x-ray emission as reconnection progresses, although our single-temperature equilibrium spectral models are not able to provide adequate fits to all the SXR data.

ATP hydrolysis in human Eg5 kinesin monitored by millisecond time‐resolved FTIR: real‐time dynamics of conformation and chemistry in vitro

Human Eg5 (HsEg5), a kinesin motor protein, is capable of ATP hydrolysis and utilizes the energy to move along the microtubule and participate in the spindle pole segregation during mitosis in eukaryotic cells. Herein, we present time‐lapsed monitoring of an in vitro ATP hydrolysis reaction by HsEg5 with ms time resolution. To survey dynamic structural and chemical changes during ATP hydrolysis concomitantly, difference Fourier‐transform infrared spectroscopy was used to monitor HsEg5 catalysis, by triggering initiation of the reaction by UV‐photolysis of caged ATP. A HPO3− vibrational mode was observed in our time‐lapse infrared data at 10 s after ATP release. This mode is assigned to formation of Pi by HsEg5, consistent with steady‐state measurement of enzyme kinetics (Vmax = 0.11 s−1). In the carbonyl stretching, or amide I, vibration of the peptide backbone in the same time‐lapse spectra, four modes were observed, reflecting net changes that occur in the secondary structure of Eg5 in a temporal sequence. These amide I modes indicate four unique conformational changes, or 'switches,' were detected in HsEg5 catalysis that may correspond to different intermediates, such as ATP binding, isomerization, and bond cleavage. Moreover, there is a subset of conformational changes that precede Pi release and there is an independent set of conformational changes as occur in tandem with Pi release.

Lipid Mixing and Content Release in Single-Vesicle, SNARE-driven Fusion Assay with 5 ms Time Resolution

Lipid Mixing and Content Release in Single-Vesicle, SNARE-driven Fusion Assay with 5 ms Time Resolution

Structural Changes in the Myosin Motors During Activation of Skeletal Muscle

Structural changes in the myosin motors during the transition from the resting state to the plateau of isometric contraction were investigated by X-ray interference from single fibers of frog skeletal muscle. Isolated intact fibers (2.1μm sarcomere length, 4°C) were mounted vertically at beamline ID2 of the ESRF synchrotron (Grenoble, France) between a loudspeaker motor and a capacitance force transducer. 2D diffraction patterns were collected on a CCD detector 10 m from the preparation with 5 ms time resolution. During the development of the isometric tetanus, the intensity of the M3 reflection, originating from the axial repeat of the myosin motors, first decreases to 30% (at 50 ms) of its resting value, then increases to a steady value 70% of that at rest. The M3 reflection has a major peak with spacing 14.34 nm at rest and two peaks with mean spacing 14.57 nm at the tetanus plateau (Linari et al., Proc. Natl. Acad. Sci. USA 97:7226, 2000). The changes in the fine structure of the M3 reflection during activation were best fit by a structural model in which (1) all thick filaments have the same mean spacing at a given time during activation (2) the number of active motors increases in proportion to the isometric force (Brunello et al., J. Physiol. 577:971 2006), (3) the conformation of the active motors is independent of the level of force and strain in the thick filament.Supported by MiUR Italy, MRC UK, CNISM, EMBO, ESRF, EMBL.