Ms Time Resolution Research Articles

Probing membrane proteins: Monitoring proton-translocation quantitatively

Proton-translocating membrane-bound proteins create and maintain pH gradients across membranes, playing important bioenergetic and regulatory roles. Some of these proteins are implied in ageing and neurodegeneration, others in tumor biology and drug resistance. Unfortunately, membrane proteins are notoriously difficult to study. They are vastly underrepresented in the growing body of protein structures, and recent proteomics advances have mostly involved soluble proteins.We present a system for quantitatively monitoring proton-translocation by membrane proteins. The protein is reconstituted into large vesicles, which provide a well defined environment. Optionally, the protein can be first fused to a cytochrome, facilitating unidirectional reconstitution1. The pH inside and outside the vesicles is monitored simultaneously using novel membrane-impermeable nanoprobes2,3. Using CCD detection, full spectral coverage of the nanoprobes and any protein-bound chromophores is delivered at up to ms time resolution. The proteoliposomes are then subjected to computer-controlled, semi-automated titrations of substrate and/or pH changes4.Key improvements over similar methodologies previously employed are the advantageous characteristics of the nanoprobes, the precision and speed of the titration system, and the CCD detection technology. Combined, these features ensure robust, fast, quantitative results on proton-pumping stoichiometries across membranes.Using this method, we characterize the lipid bilayer of the vesicles in terms of passive ion leakage and stability. We then investigate subunits of respiratory Complex I, and their antiporter homologues, reconstituted into liposomes (see Sindra Peterson Arskold's poster).1. Gustavsson, T., Eek, M., Leiding, T., Peterson Arskold, S., and Hagerhall, C. (2008) Submitted to Biochimica et Biophysica Acta Bioenergetics.2. Finikova, O., Galkin, A., Rozhkov, V., Cordero, M., Hagerhall, C., and Vinogradov, S. (2003) J. Am. Chem. Soc. 125, 4882-4893.3. Leiding, T., Gorecki, K., Vinogradov, S., Hagerhall, C., and Peterson Arskold, S. (2008) Submitted to Analytical Biochemistry.4. Autonomous Science Machines TM.

Laser scanning microscope for scanning along 3D trajectories with 5 ms time resolution

Event Abstract Back to Event Laser scanning microscope for scanning along 3D trajectories with 5 ms time resolution Gergely Katona1*, Balazs Chiovini1, Szilveszter E. Vizi1 and Balazs Rozsa1 1 Institute of Experimental Medicine Hungarian Academy of Sciences, Hungary We developed a new scanning strategy and a new 3D two-photon microscope for dendritic imaging with 5 ms time resolution. We use our multiple line scan technique (Differential distribution of NCX1 contributes to spine-dendrite compartmentalization in CA1 pyramidal cells (2007) Proc. Natl. Acad. Sci. USA, 10.1073) with the combination of two z-scanning methods. This combination results in movement typical for a roller coaster. Our technique provides 20 µm scanning range along axis z when it is used with 6 ms repetition rate. The spatial resolution (namely the mean half width of the point spread function) is better than 500 nm. We optimized our detectors for scattered photons; therefore, our system provides an excellent signal to noise ratio when compared to commercially available systems. Using our method, we measured the mechanism of the initiation and spread of dendritic regenerative activity (dendritic spikes) induced by focal electric stimulation. Our technique revealed a new form of dendritic spikes and synaptic integration in hippocampal CA1 stratum radiatum interneurons. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Research on the cerebral cortex and related structures Citation: Katona G, Chiovini B, Vizi SE and Rozsa B (2009). Laser scanning microscope for scanning along 3D trajectories with 5 ms time resolution. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.204 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Mar 2009; Published Online: 06 Mar 2009. * Correspondence: Gergely Katona, Institute of Experimental Medicine Hungarian Academy of Sciences, Budapest, Hungary, katonag@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gergely Katona Balazs Chiovini Szilveszter E Vizi Balazs Rozsa Google Gergely Katona Balazs Chiovini Szilveszter E Vizi Balazs Rozsa Google Scholar Gergely Katona Balazs Chiovini Szilveszter E Vizi Balazs Rozsa PubMed Gergely Katona Balazs Chiovini Szilveszter E Vizi Balazs Rozsa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.

Intracloud discharge and the correlated basic charge structure of a thunderstorm in Zhongchuan, a Chinese Inland Plateau region

The intracloud (IC) discharge is closely related to the charge structure of thunderstorms. The location, charge moment and polarity of intracloud discharges have been analyzed by using the electric field changes from a 7-site network of slow antennas synchronized by GPS with 1 μs time resolution in the area of Zhongchuan in the Chinese Inland Plateau. Ten IC flashes, from a storm on August 20, 2004, have been fitted by using the non-linear least-square solution. The results show that five IC flashes occurred between the main negative charge region and the lower positive charge region and other five between the main negative charge region and the upper positive charge region during the mature stage of the thunderstorm. The centers of discharge were 3.2–5.6 km and 6.8–7.7 km above sea level. The neutralized moments were about 4.56–61.0 C km and 1.06–15.9 C km. It suggests that the charge structure related to the lightning discharge can be represented by a tripole but with a strong positive charge region in the lower part of the thunderstorm, with the lower positive charge region taking an active role in the discharge.

X-ray Kinematography of Phase Transformations of Three-Component Lipid Mixtures: A Time-Resolved Synchrotron X-ray Scattering Study Using the Pressure-Jump Relaxation Technique

By using the pressure-jump relaxation technique in combination with time-resolved synchrotron small-angle X-ray diffraction (TRSAXS), the kinetics of lipid phase transformations of ternary lipid mixtures serving as model systems of heterogeneous raftlike membranes were investigated. To this end, we first established the temperature-pressure phase diagram of a model lipid raft mixture, 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC)/1,2-dipalmitoyl- sn-glycero-3-phosphatidylcholine (DPPC)/cholesterol (1:2:1), using Fourier transform infrared spectroscopy and SAXS, covering the pressure range from 1 bar to 10 kbar at temperatures in the range from 7 to 80 degrees C. We then studied the kinetics of interlamellar phase transitions of the ternary lipid system involving transitions from the fluidlike (liquid-disordered, l d) phase to the liquid-ordered (l o)/liquid-disordered (l d) two-phase coexistence region as well as between the two- and three-phase coexistence regions of the system, where also solid-ordered phases (s o) are involved. The phase transition from the all-fluid l d phase to the l o+l d two-phase coexistence region turns out to be rather rapid. Phases appear or disappear within the 25 ms time resolution of the technique, followed by a slow lattice relaxation process, which, depending on the pressure-jump amplitude, takes several seconds. Contrary to many one-component phospholipid phase transitions, the kinetics of the l d <--> l o+l d transition follows a similar time scale and mechanism for the pressurization and depressurization direction. A similar behavior is observed for the phase transition kinetics of the s o+l o+l d <--> l o+l d transformation and even for the s o+l o+l d <--> l d transformation, jumping across the l o+l d two-phase region. All transitions are fully reversible, and no intermediate states are populated. As indicated by the complex relaxation profiles observed, the overall rates observed seem to reflect the effect of coupling of various dynamical processes through the transformation, involving fast conformational changes in the sub-millisecond time regime and slow relaxation of the lattices growing, probably being largely controlled by the transport and redistribution of water into and in the new phases of the multilamellar vesicle assemblies.

A New Method for the Time-Resolved Analysis of Structure and Orientation: Polarization Modulation Infrared Structural Absorbance Spectroscopy

Polarization modulation infrared linear dichroism (PM-IRLD) is often used for measurements of molecular orientation with high sensitivity and good time resolution. However, PM-IRLD is unable to provide the structural absorbance spectrum because it does not measure separately the parallel and perpendicular spectra. Here we propose a new method, named polarization modulation infrared structural absorbance spectroscopy (PM-IRSAS), to overcome this limitation of PM-IRLD. PM-IRSAS measures the dichroic difference and structural absorbance spectra simultaneously and, therefore, allows quantitative analysis of molecular orientation and conformation with 200 ms time resolution. The PM-IRSAS method was first validated through comparison with conventional polarized FT-IR spectroscopy using drawn polymer films. Second, it was demonstrated that the PM-IRSAS method can provide a quantitative analysis of dynamic orientation and conformation changes in PET films during deformation and crystallization processes.

Covalent labeling of functional states of the acetylcholine receptor. Effects of antagonists on the receptor conformation.

Photoaffinity labeling of membrane-bound nicotinic acetylcholine receptor from Torpedo marmorata electric tissue with the ion-channel blocker [3H]TPMP+ reveals various functional states of the receptor protein if labeling is performed with ms time resolution. In the resting and in the activated state most of the label is incorporated into the alpha-polypeptide chains of the receptor complex. When equilibrated with agonists and antagonists, predominantly the delta-polypeptide chain (and to a lesser extent the beta-chain) reacts with the photolabel. Reactivity of the delta-chain increases after exposure to cholinergic effectors with a half-life slower than the kinetics of receptor activation or rapid desensitization. Agonists and antagonists stimulate photolabelling of the delta-chain with different kinetics. For acetylcholine, carbamoylcholine and suberyldicholine the half-life of the reactivity increases is 400 - 500 ms; for the antagonists hexamethonium, d-tubocurarine and flaxedil it is about 10 s. The latter slow kinetics are also observed when the receptor is preequilibrated with agonists or antagonists prior to mixing with [3H]TPMP+ and starting the photoreaction. We conclude that time-resolved photoaffinity labeling can convalently mark protein structures involved in receptor functions. Of special interest is the observation that antagonists also induce a conformational change in the receptor protein.

Simultaneous absolute timing of the Crab pulsar at radio and optical wavelengths

Context. The Crab pulsar emits across a large part of the electromagnetic spectrum. Determining the time delay between the emission at different wavelengths will allow to better constrain the site and mechanism of the emission. We have simultaneously observed the Crab Pulsar in the optical with S-Cam, an instrument based on Superconducting Tunneling Junctions (STJs) with μs time resolution and at 2 GHz using the Nancay radio telescope with an instrument doing coherent dedispersion and able to record giant pulses data. Aims. We have studied the delay between the radio and optical pulse using simultaneously obtained data therefore reducing possible uncertainties present in previous observations. Methods. We determined the arrival times of the (mean) optical and radio pulse and compared them using the tempo2 software package. Results. We present the most accurate value for the optical-radio lag of 255±21 μs and suggest the likelihood of a spectral dependence to the excess optical emission asociated with giant radio pulses.

Determination of torque generation from the power stroke of Escherichia coli F1-ATPase

The torque generated by the power stroke of Escherichia coli F1-ATPase was determined as a function of the load from measurements of the velocity of the γ-subunit obtained using a 0.25 µs time resolution and direct measurements of the drag from 45 to 91 nm gold nanorods. This result was compared to values of torque calculated using four different drag models. Although the γ-subunit was able to rotate with a 20× increase in viscosity, the transition time decreased from 0.4 ms to 5.26 ms. The torque was measured to be 63±8 pN nm, independent of the load on the enzyme.

Two-dimensional multiwire gas proportional detector for X-ray photon correlation spectroscopy of condensed matter

Details of a two-dimensional (2-D) multiwire gas proportional detector for X-ray photon correlation spectroscopy (XPCS) of condensed matter are described. The characteristics of the gas proportional detector at 8 keV, 0.3 pC anode charge, and 3 bar (absolute) of Xe/10%CO 2 are as follows: 8.5×10 −7 counts/s (100×100 μm 2) dark count rate, ∼μs time resolution, ∼48 and 73 μm position resolution (FWHM) along and across the anode wire direction, respectively, and ∼80% quantum efficiency. The effects of incident photon energy, anode charge (i.e., gain), gas drift depth, and gas pressure on position resolution are discussed. Static and dynamic speckle patterns, measured from disordered aerogel and polystyrene/polybutadiene blends by a partially coherent synchrotron X-ray source, demonstrate that a 2-D multiwire gas proportional detector is very suitable for the dynamic study of condensed matter with relaxation times in the order of μs to 10 3 s and atomic length scale.

A new ICP–TOFMS. Measurement and readout of mass spectra with 30 μs time resolution, applied to in-torch LA–ICP–MS

In-torch LA-ICP-MS was implemented into an in-house-built ICP-TOFMS system. The fast data acquisition capabilities of the new configuration allowed simultaneous multi-element measurement and readout of in-torch LA-ICP-MS signals with 30 micros time resolution. The measurements confirmed previously observed fine structures of in-torch generated signals and provided new insights in the dynamic processes in the plasma on a microsecond time scale. The new setup is described in detail and first figures of merit are given.

Small angle neutron scattering at very high time resolution: Principle and simulations of ‘TISANE’

The time resolution of SANS experiments is generally limited by frame overlap to some ms. We report on a new time-resolved stroboscopic SANS method, called TISANE, offering μs time resolution without a major sacrifice in intensity by making use of very large frame overlap. We may explore a new field in neutron scattering and complement the emerging field of time resolved small angle X-ray scattering. Here we discuss the principle of TISANE, its mathematical treatment and its limitations.

Flow Dynamics and Plasma Heating of Spheromaks in SSX

We report several new experimental results related to flow dynamics and heating from single dipole-trapped spheromaks and spheromak merging studies at SSX. Single spheromaks (stabilized with a pair of external coils, see Brown, Phys. Plasmas 13 102503 (2006)) and merged FRC-like configurations (see Brown, Phys. Plasmas 13, 056503 (2006)) are trapped in our prolate (R = 0.2 m, L = 0.6 m) copper flux conserver. Local spheromak flow is studied with two Mach probes (r 1 ≤ ρ i , r 2 ≥ ρ i ) calibrated by time-of-flight with a fast set of magnetic probes at the edge of the device. Both Mach probes feature six ion collectors housed in a boron nitride sheath. The larger Mach probe will ultimately be used in the MST reversed field pinch. Line averaged flow is measured by ion Doppler spectroscopy (IDS) at the midplane. The SSX IDS instrument measures with 1 μs or better time resolution the width and Doppler shift of the C III impurity (H plasma) 229.7 nm line to determine the temperature and line-averaged flow velocity (see Cothran, RSI 77, 063504 (2006)). We find axial flows up to 100 km/s during formation of the dipole trapped spheromak. Flow returns at the wall to form a large vortex. Recent high-resolution IDS velocity measurements during spheromak merging show bi-directional outflow jets at ±40 km/s (nearly the Alfven speed). We also measure T i ≥ 80 eV and T e ≥ 20 eV during spheromak merging events after all plasma facing surfaces are cleaned with helium glow discharge conditioning. Transient electron heating is inferred from bursts on a four-channel soft x-ray array. The spheromaks are also characterized by a suite of magnetic probe arrays for magnetic structure B(r,t), and interferometry for n e . Finally, we are designing a new oblate, trapezoidal flux conserver for FRC studies. Equilibrium and dynamical simulations suggest that a tilt-stable, oblate FRC can be formed by spheromak merging in the new flux conserver.

Three-Dimensional Particle Tracking via Bifocal Imaging

We introduce a bifocal imaging method that enables three-dimensional (3D) tracking of both fluorescent and nonfluorescent particles. We accomplish this by simultaneously imaging a focused plane, for in-plane position (x,y), and a defocused plane, for out-of-plane position (z), of a molecule using a single CCD camera. We applied our method to several systems including in vivo melanosome tracking and phagocytosed fluorescent bead tracking. We have achieved 2-5 nm accuracy with a 2-50 ms time resolution.

Probing in situ the Nucleation and Growth of Gold Nanoparticles by Small-Angle X-ray Scattering

We probe in situ by synchrotron SAXS/WAXS and UV-visible spectroscopy the nucleation and growth of gold nanoparticles. The use of a fast-mixing stopped-flow device enables the assessment of the whole particle formation process with a 200 ms time resolution. The number of particles, their size distribution, and the yield of the reaction is determined in real time through the quantitative analysis of the SAXS data on an absolute scale. Two ligands exhibit drastically different behaviors: when an alkanoic acid is used, a nucleation phase of 1 s is followed by a growth step whose rate is limited by the reaction of the monomers at the interface; on the other hand, when an alkylamine is used, the nucleation rate is increased by an order of magnitude, thus annealing growth by a lack of monomer and yielding R=1 nm particles in 2 s, as compared with R=3.7 nm in 12 s for the acid case.

An improved method for IEDF determination in pulsed plasmas and its application to the pulsed dc magnetron

In this paper we demonstrate an improved technique for obtaining time-resolved ion energy distribution functions (IEDFs) in pulsed plasma discharges using a commercial quadrupole mass energy analyser. The method involves extracting ions from the plasma at selected times during the pulse cycle through the application of a synchronized electrical bias on a grid assembly built in the barrel of the instrument. Placing the triggered mesh in the barrel of the energy analyser makes the ‘transit’ gap length (between the chopping mesh and neighbour electrodes) ∼2 mm and that results in a significant increase of the time resolution. Due to the high vacuum conditions inside the barrel, no unwanted ‘parasitic’ discharge will exist between the extractor and orifice. The electrostatic chopping by the built-in mesh allows time-resolved IEDFs to be determined without reference to the inherent time delay caused by the ion flight time through the spectrometer. The technique has been applied to the low-pressure pulsed dc magnetron discharge operating in Ar gas and at a pulse frequency of 100 kHz. Time-resolved IEDFs were obtained with a 1 µs time resolution, and these revealed large variations in the form and width of the ion energy spectra at different times during the discharge cycle, governed by the temporal evolution of the plasma potential. A calculation based on ion dynamics in the grid assembly agrees well with experimentally obtained results and demonstrates that this method can provide time resolutions as small as 200 ns.

Short time scale pulse stability of the Crab pulsar in the optical band

The fine structure and the variations of the optical pulse shape and phase of the Crab pulsar are studied on various time scales. The observations have been carried out on 4-m William Hershel and 6-m BTA telescopes with APD photon counter, photomultiplier based 4-channel photometer and PSD based panoramic spectrophotopolarimeter with 1 μs time resolution in 1994, 1999, 2003 and 2005–2006 years. The upper limit on the pulsar precession on Dec 2, 1999 is placed in the 10 s–2 hr time range. The evidence of a varying from set to set fine structure of the main pulse is found in the 1999 and 2003 years data. No such fine structure is detected in the integral pulse shape of 1994, 1999 and 2003 years. The drastic change of the pulse shape in the 2005–2006 years set is detected along with the pulse shape variability and quasi-periodic phase shifts.

Progression features of a stepped leader process with four grounded leader branches

The propagation features of stepped leader with four grounded branches during a negative CG flash have been analyzed by using the data from a high‐speed digital camera with 1 ms time resolution in correlation with wide‐band electric field change signatures. Four branches that initiated from a main leader trunk connected to ground in sequence and induced four separate return strokes. The propagation speed of four grounded leader branches shows an increase tendency when they progressed downward to ground. The average 2‐D speed of four grounded branches was estimated to be about 1.1 × 105 m/s. The electric field changes caused by the stepped leader were characterized by clustered pulses near the time of return stroke. Each cluster of pulses was composed of 3–4 pulses with an average time interval in a cluster of about 7.7 μs, and the time interval between two first pulses in two adjacent clusters was 30.1 μs.

A-Magnetic Optic-Mechanical Device to Quantify Finger Kinematics for fMRI Studies of Bimanual Coordination

Several fMRI studies have been performed to detect the neural correlates of stable bimanual coordination patterns in humans. Only few of those studies were accompanied by the on-line recording of the relative phase of fingers or hands, but none with high space and time resolutions. Conversely, the high-resolution recording of fingers' kinematics during fMRI would permit the quantification of the instantaneous fingers' positions, from which the instant at which transitions between different bimanual coordination patterns occur might be detected. This information could then be used to analyze fMRI data and detect the neural correlates of pattern transitions. We describe an a-magnetic optic-mechanical device (AMOMeD) able to monitor the fingers' positions during fMRI studies on bimanual coordination with 2 mm space resolution and 1 ms time resolution. From the instantaneous fingers' positions (recorded with an optical fiber system and a dedicated acquisition system), the oscillation amplitude, frequency, velocity and relative phase of fingers' are calculated. The signal from the fMRI trigger can be acquired simultaneously to synchronize the behavioral outcomes with the fMRI analysis. The results of our study show that this device does not affect fMRI signals, and that fMRI data can be processed using the simultaneous behavioral information to detect the brain areas activated during the transitions between different bimanual coordination patterns.

A CCD Spectroradiometer for Ultraviolet Actinic Radiation Measurements

Abstract A new spectroradiometer for spectral measurements of ultraviolet (UV) atmospheric radiation (290–400 nm) using a charge coupled device (CCD) as a detector is introduced. The instrument development is motivated by the need for measurements with (a) high accuracy in the UV-B spectral range (290–315 nm) for photochemistry applications and (b) high temporal resolution in quickly changing atmospheric conditions such as partial cloud cover. The new CCD instrument is mainly intended for airborne use. It allows fast data collection (&amp;lt;300 ms time resolution for each spectrum) with improved sensitivity in the UV spectral range. The instrumental setup and its characterization in terms of stray light, dark current, noise, and detection limits are described and compared to a spectroradiometer with a photodiode array (PDA) detector. The new CCD spectroradiometer has a one order of magnitude greater sensitivity than the PDA-based spectroradiometer. However, the stray light of the CCD instrument is wavelength dependent, which requires a more complicated data evaluation procedure than the PDA instrument. Comparison with other UV spectroradiometers (a PDA spectroradiometer and two ground-based double monochromators) shows the advantages of the CCD system for UV-B measurements of actinic flux densities and photolysis frequencies of ozone and nitrogen dioxide, and the improved performance compared to PDA spectroradiometers.