Limited Time Resolution Research Articles

Excited State Intramolecular Charge Transfer Reaction in Binary Mixtures of Water and Tertiary Butanol (TBA): Alcohol Mole Fraction Dependence

The excited state intramolecular charge transfer reaction of 4-(1-azetidinyl)benzonitrile (P4C) has been studied in water-tertiary butanol (TBA) mixtures at different alcohol mole fractions by using steady state and time-resolved fluorescence spectroscopy. The ratio between the areas under the locally excited (LE) and charge transferred (CT) emission bands is found to exhibit a sharp rise at alcohol mole fraction approximately 0.04, a value at which several thermodynamic properties of this mixture is known to show anomalous change due to the enhancement of H-bonding network. The radiative rate associated with the LE emission also shows a maximum at this TBA mole fraction. Although the structural transition from the water-like tetrahedral network to the alcohol-like chain is reflected in the red shift of the absorption spectrum up to TBA mole fraction approximately 0.10, the emission bands (both LE and CT) show the typical nonideal alcohol mole fraction dependence at all TBA mole fractions. Quantum yield, CT radiative rate as well as transition moments also exhibit a nonideal alcohol mole fraction dependence. The time-resolved emission decay of P4C has been found to be biexponential at all TBA mole fractions, regardless of emission collection around either the LE or the CT bands. The time constant associated with the slow component (tau(slow)) shows a minimum at TBA mole fraction approximately 0.04, whereas such a minimum for the fast time constant, tau(fast) (representing the rate of LE --> CT conversion reaction) is not observed. The nonobservation of the minimum in tau(fast) might be due to the limited time resolution employed in our experiments.

Analysis of Kinetics in Noisy Systems: Application to Single Molecule Tethered Particle Motion

In the tethered particle motion method the length of a DNA molecule is monitored by measuring the range of diffusion of a microsphere tethered to the surface of a microscope coverslip through the DNA molecule itself. Looping of DNA (induced by binding of a specific protein) can be detected with this method and the kinetics of the looping/unlooping processes can be measured at the single molecule level. The microsphere’s position variance represents the experimental variable reporting on the polymer length. Therefore, data windowing is required to obtain position variance from raw position data. Due to the characteristic diffusion time of the microsphere, the low-pass filtering required to attain a good signal/noise ratio (S/N) in the discrimination of looped versus unlooped state impacts significantly the measurement’s time resolution. Here we present a method for measuring lifetimes based on half-amplitude thresholding and then correcting the kinetic measurements, taking into account low S/N (leading to false events) and limited time resolution (leading to missed events). This method allows an accurate and unbiased estimation of the kinetic parameters under investigation, independently of the choice of the window used for variance calculation, with potential applications to other single molecule measurements with low S/N.

Effects of Bandwidth on Observable Multipath Clustering in Outdoor/Indoor Environments for Broadband and Ultrawideband Wireless Systems

A multipath-clustering phenomenon, which is caused by the fact that scatterers tend to group together in realistic environments, has a significant impact on the channel capacity. For a band-limited system, due to the limitation of the signal time resolution, observable multipath-clustering is affected not only by propagation environments but also by the signal bandwidth. Since a larger bandwidth gives better time resolution, it is possible to observe more multipath components and, therefore, stronger clustering effects. In this paper, a new method based on the Delta-K model for multipath times of arrival (TOAs) is proposed to investigate and quantify the effect of signal bandwidth on observable multipath clustering. Furthermore, to completely characterize the time dispersion characteristics of the channel, a statistical model using a power ratio, a decay constant, and the Rician factor to describe multipath averaged power decay and amplitude fading is proposed. Newly derived formulas are given to relate the model parameters of a wideband signal, including those of the multipath TOA, averaged power decay, and amplitude fading, to those of a narrowband signal. The channel parameter estimation methods have been extensively validated by comparing the computed channel parameters with the ones extracted from the measured channel responses of 1.95 and 2.44 GHz broadband radios in metropolitan and suburban areas, and of 3-5 GHz ultrawideband signals in indoors.

A Bacterial Arginine-Agmatine Exchange Transporter Involved in Extreme Acid Resistance

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from approximately 2 ms(-1) at approximately 37 degrees C to approximately 40 ms(-1) at approximately 10 degrees C and correspond to an activation energy of approximately 14 +/- 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF.

Breaking resolution limits in ultrafast electron diffraction and microscopy

Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100-200 keV for microscopy, corresponding to speeds of 33-70% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions.

SU‐FF‐J‐96: Modeling Correlation Between External Surface Motion and Internal Organ Motion Based On 4DCT

Purpose: To develop a method for modeling the dynamic relationship between patient surface motion and internal target motion using 4DCT data. Then, to verify its capability to interpolate internal target motion with real‐time patient surface motion information. Methods and Materials: At discrete phases of a respiration cycle, the coordinates of points in a grid with specific interval on patient surface and internal target were automatically extracted from 4DCT data, then used for the experiments. A radial basis function (RBF) based neural network was employed for modeling purpose. The model was examined by eight sets of data, which consists of the coordinates of points on a patient surface and a region of interest (ROI) on either the left lung or right lung. Results: With fine sampling (grid interval < 5 voxels) of the patient surface and internal target, less than 5% interpolation error was found in seven experiments. one patient (right side of left lung) with error more than 8% and unexpected displacement more than 10 mm in 4DCT was observed. Coarse sampling (grid interval >10 voxels) will result in a larger interpolation error. In seven successful cases, the interpolation errors less than 2mm. The only failed one had interpolation error of 4 mm. Conclusion: A dynamic model is established for correlating patient surface motion with internal target motion based on 4DCT data. An acceptable interpolation accuracy is achieved for 7 of 8 cases examined in this study. The only failed one implied that for target motion with larger displacement and higher frequency, the model based on 4DCT data might be not sufficient because of its limited time resolution in acquiring those information in a respiration cycle.

Electron Multiplying Charge-Coupled Device Camera Based Fluorescence Correlation Spectroscopy

A fluorescence correlation spectroscopy (FCS) setup is built with an electron multiplying charge-coupled device camera. Although the instrument has a limited time resolution of 4 ms, compared to 0.1-0.2 mus for common instruments using avalanche photodiodes, it allows multiplexing of FCS measurements, has a software-adjustable pinhole after data collection, performs flow speed as well as flow direction measurements in microchannels and could be used to do spectral FCS. Measurements are performed on fluorescent dyes and polystyrene beads in high-viscosity media and on epidermal growth factor receptors in Chinese hamster ovary cells. Using real measurements on single spots, multiplexing of focal spots and detection elements are simulated and the results are discussed.

Structure and Reactivity of a Thermostable Prokaryotic Nitric-oxide Synthase That Forms a Long-lived Oxy-Heme Complex

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.

Fluorescence Measurements Of Expanding Strongly Coupled Neutral Plasmas

We report new detailed density profile measurements in expanding strongly coupled neutral calcium plasmas. Using laser-induced fluorescence techniques, we determine plasma densities in the range of 10(5) to 10(9) cm(-3) to with a time resolution limit as small as 7 ns. Strong coupling in the plasma ions is inferred directly from the fluorescence signals. Evidence for strong coupling at late times is presented, confirming a recent theoretical result.

Kinetic assay for measurement of enzyme concentration in situ

Metabolic pathway analysis requires accurate data regarding enzyme kinetic parameters and metabolite concentrations inside living cells. Intracellular enzyme concentration is a critical parameter that is notoriously difficult to obtain. We propose a kinetic method, based on enzyme addition, to quantify the amount of specific enzymes in metabolic pathways. The kinetic parameters known for the vast majority of enzymes were obtained in vitro with purified enzymes.More recently, with the emergence of sequenced genomes, the use of recombinant proteins as enzyme sources are now common practice, replacing the laborious purification from the original organism by the production of large amounts of protein from a suitable host such as Escherichia coli. However, this approach poses major problems regarding metabolic pathway analysis. Post-translational modifications are not likely to occur, and specific protein–protein interactions may be absent. Better strategies are clearly needed for investigating enzymes in a way more closely resembling the in vivo state. Recently, noninvasive techniques such as fluorescence spectroscopy and nuclear magnetic resonance (NMR) are assuming greater importance but still endure serious limitations [1,2]. Fluorescence spectroscopy is restricted to a few metabolites, namely NAD(P)H, or to the use of specific probes that may be introduced in a nondestructive way in living cells. In vivo NMR shows great promise but is hampered by its limited time resolution and sensitivity. Moreover, intact systems do not allow good control of individual enzymes regarding substrate concentration and reaction conditions such as pH. An alternative approach is the study of enzymes in situ [3]. This invasive technique is based on the selective cell membrane permeabilization to low-molecular weight mole-

First experimental results of time-of-flight reconstruction on an LSO PET scanner

Time-of-flight (TOF) positron emission tomography (PET) was studied and preliminarily developed in the 1980s, but the lack of a scintillator able to deliver at the same time proper time resolution and stopping power has prevented this technique from becoming widespread and commercially available. With the introduction of LSO in PET, TOF is now a feasible option. TOF reconstruction has been implemented in the CPS Hi-Rez PET scanner, both with 2D filtered-back-projection (FBP2D) and 3D ordered subset expectation maximization (OSEM3D). A new procedure has been introduced in the time alignment to compensate for the limited digital time resolution of the present electronics. A preliminary version of scatter correction for TOF has been devised and is presented. The measured time resolution of 1.2 ns (FWHM) allowed for a signal-to-noise ratio increase of about 50% in phantoms of about 40 cm transaxial size, or a gain larger than 2 in noise equivalent counts (NEC). TOF reconstruction has shown the expected improvement in SNR, both in simulation and experimental data. First experimental results show two improvements of TOF reconstruction over conventional (non-TOF) reconstruction: a lower noise level and a better capability to resolve structures deep inside large objects.A corrigendum for this article has been published in 2007 Phys. Med. Biol. 52 2335.

Reply to ‘Time-resolved sex differences in language lateralization’

In response to our meta-analysis on sex differences in language lateralization (Sommer et al ., 2004), Ortigue and colleagues point out the limited time resolution of functional MRI (fMRI), PET and echo Doppler as a possible cause for failing to find a significant sex difference in the meta-analysis. The activation pattern of fMRI and PET studies, they argue, is composed of language components, but also of non-specific cerebral activity, especially when the control task consists of passive rest. Ortigue and colleagues explain that the functionally relevant time for language is between 170 …

Comparison of Mass-Based and Non-Mass-Based Particle Measurement Systems for Ultra-Low Emissions from Automotive Sources

Drastic reduction in particle emissions of diesel-powered vehicles and new findings on the health impact of particles raise the question of a more sensitive measurement procedure. In this paper, 16 different particle mass measurement systems are compared on a diesel heavy-duty engine equipped with a particle filter to investigate their feasibility for particle characterization for future ultra-low concentration levels. The group of instruments comprises mass-related methods (filter methods, laser-induced incandescence, photoacoustic detection, photoelectric charging, combined inertial and mobility sizing, opacity) as well as non-mass-related methods (CPC, diffusion battery, diffusion charger, ELPI, light scattering). The instruments are compared on the basis of repeatability, limit of detection, sensitivity, time resolution and correlation with the regulated gravimetric filter method, and elemental carbon fraction (EC). Several time-resolved methods show good performance and give reliable results. Opacimeters and light scattering, however, reveal shortcomings at these low concentrations. For all time-resolved advanced methods, poor correlation with the regulated filter method is observed, but most of them show good correlation with the EC fraction of the particles. This outcome demonstrates the crucial role of the sampling conditions for measurement methods that do not exclude volatile material from detection. A clear improvement in sensitivity is observed when non-mass-based instruments are applied (e.g., number or surface-related methods). The results reveal that reliable measurement methods exist for future measurement procedures. However, a change in the measurement method will lead to a discontinuity in the inventories, making it difficult to compare the particle emissions from future and past vehicle generations.

Effect of Water, Methanol, and Acetonitrile on Solvent Relaxation and Rotational Relaxation of Coumarin 153 in Neat 1-Hexyl-3-methylimidazolium Hexafluorophosphate

The dynamics of solvent relaxation in ionic liquid (IL)-water, IL-methanol, and IL-acetonitrile mixtures have been investigated using steady state and picosecond time-resolved fluorescence spectroscopy. We have used Coumarin 153 (C-153) and 1-hexyl-3-methylimidazolium hexafluorophosphate ([hmim][PF(6)]) as fluorescence probe and IL, respectively. The steady-state emission spectra showed that the gradual addition of cosolvents increases the polarity of the mixtures. In neat [hmim][PF(6)] and all IL-cosolvent mixtures, solvation occurs in two well-separated time regimes within the time resolution of our instrument. A substantial portion of the solvation has been missed due to the limited time resolution of our instrument. The gradual addition of cosolvents decreases the viscosity of the medium and consequently solvation time also decreases. The decrease in solvation time is more pronounced on addition of acetonitrile compared to water and methanol. The rotational relaxation time of the probe is also decreasing with gradual addition of the cosolvents. The decrease in viscosity of the solution is responsible for the decrease in the rotational relaxation time of the probe molecule.

Detection of Intensity Change Points in Time-Resolved Single-Molecule Measurements

We present a method for the analysis of optical single molecule emission data that exhibit discrete intensity jumps. This new method uses a generalized likelihood ratio test that determines the location of an intensity change point based on individual photon arrival times. This test is applied recursively to an entire single molecule intensity trajectory, thus finding each change points. Expectation-maximization clustering and the Bayesian information criterion is then used for accurate determination of the true number of states accessible to the system. This procedure allows rigorous and quantitative determination of intensity change points without the artificial time resolution limitations that arise from binning and thresholding.

Electro-optic technique with improved time resolution for real-time, nondestructive, single-shot measurements of femtosecond electron bunch profiles.

Electro-optic detection of the Coulomb field of a relativistic electron bunch combined with single-shot cross correlation of optical pulses is used to enable single-shot measurements of the shape and length of femtosecond electron bunches. This method overcomes a fundamental time-resolution limit of previous single-shot electro-optic measurements, which arises from the inseparability of time and frequency properties of the probing optical pulse. Using this new technique we have made real-time measurements of a 50 MeV electron bunch, observing the profile of 650 fs FWHM ( approximately 275 fs rms) long bunches.

Exploring Rare Conformational Species and Ionic Effects in DNA Holliday Junctions Using Single-molecule Spectroscopy

The four-way DNA (Holliday) junction is an essential intermediate in DNA recombination, and its dynamic characteristics are likely to be important in its cellular processing. In our previous study we observed transitions between two antiparallel stacked conformations using a single-molecule fluorescence approach. The magnesium concentration-dependent rates of transitions between stacking conformers suggested that an unstacked open structure, which is stable in the absence of metal ions, is an intermediate. Here, we sought to detect possible rare species such as open and parallel conformations and further characterized ionic effects. The hypothesized open intermediate cannot be resolved directly due to the limited time resolution and sensitivity, but our study suggests that the open form is achieved very frequently, hundreds of times per second under physiologically relevant conditions. Therefore despite being a minority species, its frequent formation raises the probability that it could become stabilized by protein binding. By contrast, we cannot detect even a transient existence of the junctions in a parallel form, and the probability of such forms with a lifetime greater than 5 ms is less than 0.01%. Stacking conformer transitions are observable in the presence of sodium or hexammine cobalt (III) ions as well as magnesium ions, but the transition rates are higher for lower valence ions at the same concentrations. This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms.

Real-time monitoring of RNA and DNA reactions by fluorescence detection.

Conventional analysis of nucleic acid reactions (cleavages, ligations) is performed with a (radioactive) labeled substrate, and reaction products (one aliqout for each time-point) are analyzed by gel electrophoresis followed by detection (X-ray film + densitometer or phosphoimager). This is a cumbersome approach, and involves frequent preparation of fresh labeled substrate, and rather limited time resolution. Real-time monitoring with non-decaying fluorescent labels overcomes these problems. Two analysis methods are presented here. Fluorescence polarization with a very broad range of applications, can be used for any type of RNA or DNA conversion with significant change of mol wt (including "gel shift"). FRET (fluorescence resonance energy transfer) depends on intramolecular interaction (or in a multimolecular complex) of a fluorescent dye ("reporter") with a quencher moiety (a guanine base or a second dye). Applications include detailed kinetic studies and optimizing reactions with a wide range of conditions and highly specific nucleic acid sequence detections, challenging other hybridization-based methods. Special applications are the introduction of catalytic nucleic acids for real-time monitoring of nucleic acid amplification reactions such as NASBA and PCR.

New technique for reconstructing instantaneous velocity profiles from viscometric tests: Application to pasty materials

We present a new technique for reconstructing the instantaneous velocity profiles during creep, dynamic, or ramp tests under controlled stress in wide-gap Couette flows, from a series of similar tests under smaller stress amplitudes. This approach is based on a rigourous theory, and since it requires that the fluid does not flow close to the outer cylinder, it is particularly suitable for yield stress fluids. The interest of this reconstruction technique is that it is simpler than direct techniques (nuclear magnetic resonance, light scattering, particle imaging velocimetry, etc.) and has almost no limitations in time and space resolution. Thus, one can obtain the velocity profiles under steady-state and transient flows. We show that for a commercial hair gel the velocity profile obtained with this technique is in excellent agreement with that found from magnetic resonance imaging rheometry within the range of measurement (four decades of velocity). From other tests with a mustard and a kaolin–water suspension we demonstrate that the “viscosity bifurcation” effect observed with various pasty materials [Coussot et al., Phys. Rev. Lett. (2002a)] is directly associated with an abrupt change in the slope of the velocity profiles at the interface between the sheared and unsheared regions. We also show that the effect of wall slip on the reconstructed velocity profile is to shift the level of the unsheared region to a virtual, constant, finite, velocity level.