Limited Time Resolution Research Articles

Transient Binding and Non-rotational Coupled Motion of p53 Revealed by Sub-Millisecond Resolved Single-Molecule Fluorescence Tracking

Target specific DNA-binding proteins (DBP) are required to search for and bind to their target DNA sequence quickly and accurately using various strategies such as 1D diffusion and 3D jump. However, many more events have been postulated in the target search dynamics. For example, an encounter complex between DBP and DNA, assumed to be present before the formation of the stable DNA-DBP complex, is proposed to allow DBP to be in the proximity of DNA and promote the target search. However, the limited time resolution of the previous methods, several tens of milliseconds, hampers the distinction of these motions.

Experimental time resolution limits of modern SiPMs and TOF-PET detectors exploring different scintillators and Cherenkov emission

Solid state photodetectors like silicon photomultipliers (SiPMs) are playing an important role in several fields of medical imaging, life sciences and high energy physics. They are able to sense optical photons with a single photon detection time precision below 100 ps, making them ideal candidates to read the photons generated by fast scintillators in time of flight positron emission tomography (TOF-PET). By implementing novel high-frequency readout electronics, it is possible to perform a completely new evaluation of the best timing performance achievable with state-of-the-art analog-SiPMs and scintillation materials. The intrinsic SiPM single photon time resolution (SPTR) was measured with Ketek, HPK, FBK, SensL and Broadcom devices. Also, the best achieved coincidence time resolution (CTR) for these devices was measured with LSO:Ce:Ca of mm3 and mm3 size crystals. The intrinsic SPTR for all devices ranges between 70 ps and 135 ps FWHM when illuminating the entire mm2 or mm2 area. The obtained CTR with LSO:Ce:Ca of mm3 size ranges between 58 ps and 76 ps FWHM for the SiPMs evaluated. Bismuth Germanate (BGO), read out with state of-the-art NUV-HD SiPMs from FBK, achieved a CTR of 158 ps and 277 ps FWHM for mm3 and mm3 crystals, respectively. Other BGO geometries yielded 167 3 ps FWHM for mm3 and 235 5 ps FWHM for mm3 also coupled with Meltmount (n = 1.582) and wrapped in Teflon. Additionally, the average number of Cherenkov photons produced by BGO in each 511 keV event was measured to be 17 3 photons. Based on this measurement, we predict the limits of BGO for ultrafast timing in TOF-PET with Monte Carlo simulations. Plastic scintillators (BC422, BC418), BaF2, GAGG:Ce codoped with Mg and CsI:undoped were also tested for TOF performance. Indeed, BC422 can achieve a CTR of 35 2 ps FWHM using only Compton interactions in the detector with a maximum deposited energy of 340 keV. BaF2 with its fast cross-luminescence enables a CTR of 51 5 ps FWHM when coupled to VUV-HD SiPMs from FBK, with only ∼22% photon detection efficiency (PDE). We summarize the measured CTR of the various scintillators and discuss their intrinsic timing performance.

Urban waterlogging monitoring and early warning based on video images

Urban flood disaster causes serious loss to urban residents. Timely access to urban waterlogging conditions has great significance for disaster prevention and disaster relief. Owing to the time resolution limitation of data, the traditional monitoring of urban flood disasters using remote sensing imagery cannot realise real-time automatic monitoring and continuous monitoring of key disaster areas. This paper selects road monitoring video, uses image difference operation and support vector machine (SVM) algorithm to identify the waterlogging area, and uses the region growing method to extract the waterlogging area range. The research results show that this method can be used for continuous monitoring and early warning of urban waterlogging in real time.

Ultrafast sensing of photoconductivity decay using microwave resonators

Microwave reflectance probed photoconductivity (or μ-PCD) measurement represents a contactless method to determine the amount and lifetime of light-induced charge carriers in semiconductors. Major drawbacks of the method include a difficult separation of reflectance due to dielectric and conduction effects and that the μ-PCD signal is prohibitively weak for highly conducting samples. Both of these limitations could be tackled with the use of microwave resonators due to the well-known sensitivity of resonator parameters to minute changes in the material properties combined with a null measurement. A general misconception is that time resolution of resonator measurements is limited beyond their bandwidth by the readout electronics response time. While it is true for conventional resonator measurements, such as those employing a frequency sweep, we present a time-resolved resonator parameter readout method which overcomes these limitations and allows measurement of complex material parameters and to enhance μ-PCD signals with the ultimate time resolution limit being the resonator time constant. This is achieved by detecting the transient response of microwave resonators on the time scale of a few 100 ns during the μ-PCD decay signal. The method employs a high-stability oscillator working with a fixed frequency, which results in a stable and highly accurate measurement.

The Structure of Sub‐nm Platinum Clusters at Elevated Temperatures

AbstractLittle is known about metallic clusters consisting merely of a dozen of atoms or even less, despite of their importance in catalysis and crystal nucleation. Scanning transmission electron microscopy (STEM) provides direct atomic structure information but has inherently suffered from limited time resolution. We employ fast dynamic STEM combined with a spatio‐temporal image denoising algorithm to explore the structure and stability of Pt clusters on carbon, which represents a highly relevant catalysis system. At room temperature, dynamic amorphous 2D structures are found, while above ≈300 °C, the clusters transform into a crystalline state. Our experimental and theoretical data reveal an unexpected strong trend of the crystalline clusters to exhibit the face‐centered cubic, bulk structure of Pt with cuboidal geometries being most prominent.

The Structure of Sub-nm Platinum Clusters at Elevated Temperatures.

Little is known about metallic clusters consisting merely of a dozen of atoms or even less, despite of their importance in catalysis and crystal nucleation. Scanning transmission electron microscopy (STEM) provides direct atomic structure information but has inherently suffered from limited time resolution. We employ fast dynamic STEM combined with a spatio-temporal image denoising algorithm to explore the structure and stability of Pt clusters on carbon, which represents a highly relevant catalysis system. At room temperature, dynamic amorphous 2D structures are found, while above ≈300 °C, the clusters transform into a crystalline state. Our experimental and theoretical data reveal an unexpected strong trend of the crystalline clusters to exhibit the face-centered cubic, bulk structure of Pt with cuboidal geometries being most prominent.

Relaxation Dispersion NMR to Reveal Fast Dynamics in Brønsted Acid Catalysis: Influence of Sterics and H-Bond Strength on Conformations and Substrate Hopping.

NMR provides both structural and dynamic information, which is key to connecting intermediates and to understanding reaction pathways. However, fast exchanging catalytic intermediates are often inaccessible by conventional NMR due its limited time resolution. Here, we show the combined application of the 1H off-resonance R1ρ NMR method and low temperature (185–175 K) to resolve intermediates exchanging on a μs time scale (ns at room temperature). The potential of the approach is demonstrated on chiral phosphoric acid (CPA) catalysts in their complexes with imines. The otherwise inaccessible exchange kinetics of the E-I ⇌ E-II imine conformations and thermodynamic E-I:E-II imine ratios inside the catalyst pocket are experimentally determined and corroborated by calculations. The E-I ⇌ E-II exchange rate constants (kex185 K) for different catalyst–substrate binary complexes varied between 2500 and 19 000 s–1 (τex = 500–50 μs). Theoretical analysis of these exchange rate constants revealed the involvement of an intermediary tilted conformation E-III, which structurally resembles the hydride transfer transition state. The main E-I and E-II exchange pathway is a hydrogen bond strength dependent tilting–switching–tilting mechanism via a bifurcated hydrogen bond as a transition state. The reduction in the sterics of the catalyst showed an accelerated switching process by at least an order of magnitude and enabled an additional rotational pathway. Hence, the exchange process is mainly a function of the intrinsic properties of the 3,3′-substituents of the catalyst. Overall, we believe that the present study opens a new dimension in catalysis via experimental access to structures, populations, and kinetics of catalyst–substrate complexes on the μs time scale by the 1H off-resonance R1ρ method.

Two-site versus continuum diffusion model of blocker dynamics in a membrane channel: Comparative analysis of escape kinetics

This paper deals with the escape of a charged blocker molecule from a voltage-biased membrane channel with a constriction zone which the molecule cannot pass through. Recently, we developed a two-site model of the blocker dynamics in such a system and applied it to analyze the escape kinetics. Here, we compare the decay of the blocker survival probability predicted by the two-site model with that for the survival probability given by a more accurate model which assumes continuum diffusion of the blocker molecule inside the channel. The main finding of the present work is that both models predict the same decay of the survival probability at long times. This result is of practical importance since only the long-time tail of the survival probability can be studied in single-channel experiments in which, due to a limited time resolution, only long-lasting individual events of channel blockades can be measured.

Membrane potential manipulation with visible flash lamp illumination of targeted microbeads

The electrical membrane potential (Vm) is a key dynamical variable of excitable membranes. Despite the tremendous success of optogenetic methods to modulate Vm with light, there are some shortcomings, such as the need of genetic manipulation and limited time resolution. Direct optical stimulation of gold nanoparticles targeted to cells is an attractive alternative because the absorbed energy heats the membrane and, thus, generates capacitive current sufficient to trigger action potentials [1, Carvalho-de-Souza et al., 2015]. However, focused laser light is required and precise location and binding of the nanoparticles cannot be assessed with a conventional microscope. We therefore examined a complementary method to manipulate Vm in a spatio-temporal fashion by non-focused visible flashlight stimulation (Xenon discharge lamp, 385-485 nm, ∼500 μs) of superparamagnetic microbeads. Flashlight stimulation of single beads targeted to cells resulted in transient inward currents under whole-cell patch-clamp control. The waveform of the current reflected the first time derivative of the local temperature induced by the absorbed light and subsequent heat dissipation. The maximal peak current as well as the temperature excursion scaled with the proximity to the plasma membrane. Transient illumination of light-absorbing beads, targeted to specific cellular sites via protein-protein interaction or direct micromanipulation, may provide means of rapid and spatially confined heating and electrical cell stimulation.

Photocalorespirometry (Photo-CR): A Novel Method for Access to Photosynthetic Energy Conversion Efficiency

One key parameter for assessing the CO2 fixation in aquatic ecosystems but also for the productivity of photobioreactors is the energy conversion efficiency (PE) by the photosynthetic apparatus. PE strictly depends on a range of different fluctuating environmental conditions and is therefore highly variable. PE is the result of complex metabolic control. At the moment PE can only be determined indirectly. Furthermore, the currently available techniques either capture only short time processes, thus reflecting only parts of the photosynthetic engine, or quantify the total process but only with limited time resolution. To close this gap, we suggest for the first time the direct measurement of the fixed energy combined with respirometry, called photocalorespirometry (Photo-CR). The proof of the principle of Photo-CR was established with the microalga Chlamydomonas reinhardtii. The simultaneous measurement of oxygen production and energy fixation provides an calorespirometric ratio of −(437.9 ± 0.7) kJ mol−1 under low light conditions. The elevated calorespirometric ratio under high light conditions provides an indication of photo-protective mechanisms. The Photo-CR delivers the PE in real time, depending on the light intensity. Energetic differences less than 0.14% at radiation densities of up to 800 μE m−2 s−1 can be quantified. Other photosynthetic growth parameters (e.g. the specific growth rate of 0.071 h−1, the cell specific energy conservation of 30.9 ± 1.3 pW cell−1 at 150 µE m−2 s−1 and the number of photons (86.8) required to fix one molecule of CO2) can easily be derived from the Photo-CR data.

High-frequency SiPM readout advances measured coincidence time resolution limits in TOF-PET

Scintillator based radiation detectors readout by SiPMs successively break records in their reached time resolution. Nevertheless, new challenges in time of flight positron emission tomography (TOF-PET) and high energy physics are setting unmatched goals in the 10 ps range. Recently it was shown that high frequency (HF) readout of SiPMs significantly improves the measured single photon time resolution (SPTR), allowing to evaluate the intrinsic performance of large area devices; e.g. FBK NUV-HD SiPMs of mm2 area and 40 m single photon avalanche diode (SPAD) size achieve 90 ps FWHM. In TOF-PET such readout allows to lower the leading edge detection threshold, so that the fastest photons produced in the crystal can be utilized. This is of utmost importance if a high SPTR and prompt Cherenkov light generated by the hot-recoil electron upon 511 keV photo-absorption should improve timing. This paper shows that high-frequency bipolar transistor readout of state-of-the-art SiPMs coupled to high-performance scintillators can substantially improve the best achievable coincidence time resolution (CTR) in TOF-PET. In this context a CTR of 158 3 ps FWHM with mm3 BGO crystals coupled to FBK SiPMs is achieved. This faint Cherenkov signal is as well present in standard LSO scintillators, which together with low SPTR values (<90 ps FWHM) improves the CTR of mm3 LSO:Ce:Ca coupled to FBK NUV-HD mm2 with 25 m SPAD size to 61 2 ps FWHM using HF-electronics, as compared to 73 2 ps when readout by the NINO front-end ASIC. When coupling the LSO:Ce:Ca crystals to FBK NUV-HD SiPMs of mm2 and 40 m SPAD size, using HF-electronics, a CTR of even 58 3 ps for mm3 and 98 3 ps for mm3 is achieved. This new experimental data will allow to further discuss the timing limits in scintillator-based detectors.

Noisy defects in the high-Tc superconductor Bi2Sr2CaCu2O8+x

Dopants and impurities are crucial in shaping the ground state of host materials: semiconducting technology is based on their ability to donate or trap electrons, and they can even be used to transform insulators into high temperature superconductors. Due to limited time resolution, most atomic-scale studies of the latter materials focussed on the effect of dopants on the electronic properties averaged over time. Here, by using atomic-scale current-noise measurements in optimally doped Bi2Sr2CaCu2O8+x, we visualize sub-nanometre sized objects where the tunnelling current-noise is enhanced by at least an order of magnitude. We show that these objects are previously undetected oxygen dopants whose ionization and local environment leads to unconventional charge dynamics resulting in correlated tunnelling events. The ionization of these dopants opens up new routes to dynamically control doping at the atomic scale, enabling the direct visualization of local charging on e.g. high-Tc superconductivity.

Inline observation of tool wear in deep drawing with thermoelectric and optical measurements

Wear is an industrially relevant process limit in sheet metal forming. So far, detection of wear in these processes is based on offline analyses of tool surfaces. This procedure leads to wear data with a limited resolution in time. Since the restricted data acquisition increases the probability of scrap production and limits the building of improved wear models, higher resolutions of wear data are needed. Results derived in strip drawing tests reveal that wear data from thermoelectric current and sheet topography measurements can serve as appropriate basis for condition monitoring of forming tools and wear model set-up.

Investigation of the photoinduced axial ligation process in the excited state of nickel(II) phthalocyanine

Photoinduced axial ligation process in the excited state of nickel(II) octabutoxyphthalocyanine (NiPcOBu8) in pyridine solvent has been investigated by ultrafast optical transient absorption and X-ray transient absorption spectroscopies. Although Ni phthalocyanine complexes have been reported to have no tendency to form six-coordinate structure, the (dz2,dx2-y2) state of NiPcOBu8 generated by photoexcitation of Q-band was discovered to axially ligate with the pyridine molecule very rapidly, on a timescale of ˜100 ps. Although the presence of an excited unligated (d,d) state cannot be confirmed due to the time resolution limit of the synchrotron X-ray probe pulse with ˜80 ps FWHM, the biexponential kinetics of the ground state recovery detected by the 1s → 4pz feature suggests the decay of the unligated (d,d) species with an 82 ps time constant, while that of the ligated NiPcOBu8 species is 3–5 ns. The photoinduced ligation dynamics of NiPcOBu8 is vastly different compared to that of Ni porphyrin complexes. This study provides a structural basis on the ligation chemistry in both the ground and excited states of nickel complexes based on their electronic and nuclear structures.

Topological structures are consistently overestimated in functional complex networks

Functional complex networks have meant a pivotal change in the way we understand complex systems, being the most outstanding one the human brain. These networks have classically been reconstructed using a frequentist approach that, while simple, completely disregards the uncertainty that derives from data finiteness. We provide here an alternative solution based on Bayesian inference, with link weights treated as random variables described by probability distributions, from which ensembles of networks are sampled. By using both statistical and topological considerations, we prove that the role played by links’ uncertainty is equivalent to the introduction of a random rewiring, whose omission leads to a consistent overestimation of topological structures. We further show that this bias is enhanced in short time series, suggesting the existence of a theoretical time resolution limit for obtaining reliable structures. We also propose a simple sampling process for correcting topological values obtained in frequentist networks. We finally validate these concepts through synthetic and real network examples, the latter representing the brain electrical activity of a group of people during a cognitive task.

Cryofixation during live-imaging enables millisecond time-correlated light and electron microscopy.

Researchers seek to link cellular functions to their smallest structural components. Currently this requires correlation of two imaging techniques, live imaging and electron microscopy. Current correlative methods, however, have limited time resolution due to the sample preparation procedures for electron microscopy. Following live imaging, samples are transferred from the light microscope to a cryofixation, or ultra-fast freezing, instrument. The biological process progresses until the sample freezes, 1 second or more after the last live image. In this work, samples are cryofixed directly within the light microscope field of view. By eliminating the transfer step, time correlation between light and electron microscopy images of our samples is limited only by the freezing rate to the order of milliseconds rather than seconds.

Coarse grained quantum dynamics

Inspired by holographic Wilsonian renormalization, we consider coarse graining a quantum system divided between short distance and long distance degrees of freedom, coupled via the Hamiltonian. Observations using purely long distance observables are described by the reduced density matrix that arises from tracing out the short-distance degrees of freedom. The dynamics of this density matrix is non-Hamiltonian and nonlocal in time, on the order of some short time scale. We describe this dynamics in a model system with a simple hierarchy of energy gaps $\Delta E_{UV} > \Delta E_{IR}$, in which the coupling between high-and low-energy degrees of freedom is treated to second order in perturbation theory. We then describe the equations of motion under suitable time averaging, reflecting the limited time resolution of actual experiments, and find an expansion of the master equation in powers of $\Delta E_{IR}/\Delta E_{UV}$, after the fashion of effective field theory. The failure of the system to be Hamiltonian or even Markovian appears at higher orders in this ratio. We compute the evolution of the density matrix in three specific examples: coupled spins, linearly coupled simple harmonic oscillators, and an interacting scalar QFT. Finally, we argue that the logarithm of the Feynman-Vernon influence functional is the correct analog of the Wilsonian effective action for this problem.

A pico-second resolution arbitrary timing generator based on time folding and time interpolating.

We report a pico-second resolution arbitrary timing generator which is implemented with a field-programmable-gate-array. The arbitrary timing/pattern generator is based on a time folding method which is combined with a delay chain for fine time interpolating. The time folding method can not only break the limitation of sequence time resolution contributed by the minimum chain cell delay but also improve the chain linearity. The arbitrary timing generator which is based on the time folding technique is integrated in a printed-circuit board, and a 5 ps time resolution with enhanced output linearity is obtained. The dynamic range of output pulses from the arbitrary timing generator is from 5 ns to 10 s. In this paper, we describe the principle, the circuit design, and the characterizations of the arbitrary timing generator. We also discuss the improvement of performance in timing generation using the time folding method. The high-performance arbitrary timing generator has a bright future to be used in the applications that require high-resolution timing sequence generation.

Brain Monitoring of Sedation in the Intensive Care Unit Using a Recurrent Neural Network.

Over and under-sedation are common in critically ill patients admitted to the Intensive Care Unit. Clinical assessments provide limited time resolution and are based on behavior rather than the brain itself. Existing brain monitors have been developed primarily for non-ICU settings. Here, we use a clinical dataset from 154 ICU patients in whom the Richmond Agitation-Sedation Score is assessed about every 2 hours. We develop a recurrent neural network (RNN) model to discriminate between deep vs. no sedation, trained end-to-end from raw EEG spectrograms without any feature extraction. We obtain an average area under the ROC of 0.8 on 10-fold cross validation across patients. Our RNN is able to provide reliable estimates of sedation levels consistently better compared to a feed-forward model with simple smoothing. Decomposing the prediction error in terms of sedatives reveals that patient-specific calibration for sedatives is expected to further improve sedation monitoring.

Communication: Transition-path velocity as an experimental measure of barrier crossing dynamics.

Experimental observation of transition paths-short events when the system of interest crosses the free energy barrier separating reactants from products-provides an opportunity to probe the dynamics of barrier crossing. Yet limitations in the experimental time resolution usually result in observing trajectories that are smoothed out, recross the transition state fewer times, and exhibit apparent velocities that are much lower than the instantaneous ones. Here we show that it is possible to define (and measure) an effective transition-path velocity which preserves exact information about barrier crossing dynamics in the following sense: the exact transition rate can be written in a form resembling that given by transition-state theory, with the mean thermal velocity replaced by the transition-path velocity. In addition, the transition-path velocity (i) ensures the exact local value of the unidirectional reactive flux at equilibrium and (ii) leads to the exact mean transition-path time required for the system to cross the barrier region separating reactants from products. We discuss the coordinate dependence of the transition path velocity and derive analytical expressions for it in the case of diffusive dynamics. These results can be used to discriminate among models of barrier crossing dynamics in single-molecule force spectroscopy studies.