Time-resolved Recording Research Articles

Time-Resolved Confocal Fluorescence Microscopy: A Generalized Approach Enables New Directions for FLIM, FRET and FCS

Fluorescence dynamics of single molecules can be followed on timescales from sub-nanoseconds to seconds and even beyond with a universal approach of time-resolved measurements. The underlying technique (Time-Tagged Time-Resolved (TTTR) Recording) allows one to simultaneously record timing and fluorescence intensity information, both spectrally and spatially, on a single photon basis. We apply photon sorting and weighting schemes determined from the nanosecond photon arrival times to extend and improve single-molecule fluorescence methodologies which up to now commonly utilize only intensity-based analysis, namely FCS and FRET.In Fluorescence Lifetime Correlation Spectroscopy (FLCS) photon weighting provides superior suppression of common parasitic contributions, e.g., Raman scattering and detector after-pulsing. Beyond this improvement of traditional FCS, FLCS also offers the possibility to accurately determine diffusion properties of different species only requiring that the species differ in their fluorescence lifetimes [1]. In 2-focus-FCS (2fFCS), the nanosecond timing information is used to identify the spatial origin of the photons by combining Pulsed Interleaved Excitation (PIE) with time-gated detection [2]. Thereby, 2fFCS dramatically improves the accuracy of measuring absolute diffusion coefficients. In addition to this, PIE can be used to identify artifacts and sub-populations in single-pair FRET measurements. Nanosecond time-resolved detection offers a complementary approach to donor/acceptor intensity based methods for calculating FRET efficiencies via quenching of the donor lifetime.[1] Benda A., Hof. M., Wahl M., Patting M., Erdmann R., Kapusta P., Rev. Sci. Instr., Vol.76, 033106 (2005)[2] Dertinger Th., Pacheco V., von der Hocht I., Hartmann R., Gregor I., Enderlein J, ChemPhysChem, Vol.8, p.433 (2007)

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.

Measurement of water film thickness by laser-induced fluorescence and Raman imaging

We present two non-intrusive, laser-based imaging techniques for the quantitative measurement of water fluid film thickness. The diagnostics methods are based on laser-induced fluorescence (LIF) of the organic tracer ethyl acetoacetate added to the liquid in sub-percent (by mass) concentration levels, and on spontaneous Raman scattering of liquid water, respectively, both with excitation at 266 nm. Signal intensities were calibrated with measurements on liquid layers of known thickness in a range between 0 and 500 μm. Detection via an image doubler and appropriate filtering in both light paths enabled the simultaneous detection of two-dimensional liquid film thickness information from both methods. The thickness of water films on transparent quartz glass plates was determined with an accuracy of 9% for the tracer LIF and 15% for the Raman scattering technique, respectively. The combined LIF/Raman measurements also revealed a preferential evaporation of the current tracer during the time-resolved recording of film evaporation.

Monitoring Sol-to-Gel Transitions via Fluorescence Lifetime Determination Using Viscosity Sensitive Fluorescent Probes

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials.

Force-Generating Cross-Bridges during Ramp-Shaped Releases: Evidence for a New Structural State

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5°C, force transients were slowed sufficiently to record diffraction patterns in several 2–4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATP γS] and at 1°C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.

Controlling a Single Protein in a Nanopore through Electrostatic Traps

Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.

Surface-bound chemical vapour deposition of carbon nanotubes: In situ study of catalyst activation

We combine in situ X-ray photoelectron spectroscopy and ex situ electron and Raman spectroscopy to study chemical interactions of SiO 2-supported Fe catalyst films during C 2H 2 exposure in the 400–600 °C temperature range. Carbon nanotubes nucleate at C 2H 2 pressures below 10 −6 mbar, which allows a time-resolved recording of core level spectra. We find a rapid transition from an initial chemisorbed carbon on metallic Fe catalyst nanoparticles to a sp 2 graphitic carbon network. Pre-treating the Fe catalyst film by H 2 sputtering influences the initial catalyst de-wetting and increases carbon nanotube yield.

Implosion of a spherical shock wave reflected from a spherical wall

The paper describes results of experiments of a converging spherical shock wave reflected from a spherical wall. In order to visualize the motion and the flow field behind the shock waves, an aspheric lens-shaped transparent test section made of acrylic PMMA (polymethyl methacrylate) with an inner spherical cavity was designed and constructed. This test section made optical flow visualization with collimated object beams possible. Spherical shock waves were produced at the centre of the spherical cavity by explosion of silver azide pellets ranging from 1.0 to 10.0 mg with corresponding energies of 1.9 to 19 J. The charges were ignited by irradiation of a pulsed Nd:YAG laser beam. Pressures were also measured at two points with pressure transducers mounted flush at the inner wall of the test section. The pellet was simultaneously ignited on two sides or was shaped to produce a uniform diverging spherical shock wave. This spherically diverging shock wave was reflected from the spherical inner wall of the test section to form a converging spherical shock wave. We visualized the shock-wave motion by using double exposure holographic interferometry and time-resolved high-speed video recording. The sequence of diverging and converging spherical shock-wave propagations and their interaction with gaseous explosion products were observed. The convergence, acceleration and stability of the imploding shock wave in the test section were studied.

Conformational Dynamics of Na+/K+‐ and H+/K+‐ATPase Probed by Voltage Clamp Fluorometry

We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K(+) that were completely abolished by ouabain. When voltage jumps were applied under Na(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K(+) concentrations, that is, under turnover conditions. As a result, the distribution between E(1) and E(2) states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H(+)/K(+)-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na(+)/K(+)-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.

Instabilities on a free falling jet under an internal flow breakup mode regime

Instabilities leading to the breakup of low velocity liquid jets are investigated experimentally. Pulsed shadowgraphy was used to measure local quantities on the wavy shape of the jets with an anamorphic imaging setup. Statistical recording is employed to determine the mean characteristics of the instabilities and time-resolved recording is used to follow individual waves along the jet axis. Particular attention has been paid to the changes that occur in the breakup process at the critical point of the stability curves for jets under internal flow breakup mode regime. For such jets, the critical point is not driven by aerodynamic effects and is not correctly predicted by available theories. It is shown that these jets follow the linear theory of Weber up to the critical point. The growth rate and the wave number increase steeply after the critical point. Evidence is put on the role of the relaxation of the velocity profile in the stability of the jets under internal flow breakup mode regime.

Time-Resolved Recording of Ionic Dyestuff Aggregation by Static Light Scattering

Mixtures of anionic direct dyes are used to achieve distinct color shades during dyeing of cellulose. Occasionally, the presence of bivalent alkaline-earth ions interferes with this dyeing process. A possible reason for this impairment is dyestuff aggregation. To investigate the aggregation process together with the resulting structure of the aggregates, time-resolved static light scattering was carried out with dilute solutions of a dyestuff mixture which contains 50 wt % of each component and which has an orange color shade. All scattering data of the aggregates could be interpreted consistently by means of the wormlike chain model. Comparatively short aggregates with a contour length of 250−300 nm were observed in the presence of Na2SO4 as an added salt. If the Na+ ions were exchanged by Mg2+ ions, a gradual increase of the aggregates was observed, extending over a time regime of several minutes and leading to contour lengths which were up to 10 times longer than those found in the presence of Na+ ions.

Laser-raster spectrometer for time-resolved recording of transient absorption

For measurements of transient absorption a laser-raster technique has been found to be surprisingly sensitive. It utilizes spatial separation of excitation and probing by employing a fast-flowing jet stream. The time resolution is determined by the flow velocity and the focal diameters of the continuous excitation and probing laser beams. Changes in transmission can be detected in the spectral region from 350 to 1000 nm. A dual-frequency modulation technique is used to achieve a sensitivity of 10(-4). The time resolution is 0.5 mus. The performance of this spectrometer is demonstrated by measurement of the absorption of the transient states of Rhodamine 6G dye in ethylene glycol.

New compact Echelle spectrographs with multichannel time-resolved recording capabilities

The paper describes a new type of Echelle spectrograph system that utilises a gated interline CCD camera. The system, that contains no moving parts, records simultaneously 6000 time-resolved spectral channels in the range 200–1100 nm with a lowest gate time of 100 ns and with a constant spectral resolution. Results from time-resolved recordings made both from a Xe-flash source and from laser induced breakdown, are presented. It is concluded that the gated CCD sensors are of great interest for applications in spectroscopy. They are of special interest for the application with Echelle spectrographs. The high resolution of the gatable CCD with 6.7×6.7 μm 2 surpasses that of any ICCD today and in the foreseeable future. The gated CCD also opens the NIR wavelength region for time-resolved multichannel spectroscopy.

Time-resolved studies of light propagation in paper

A method for time-resolved recording of light scattering in thin, highly scattering media is described. Subpicosecond pulses from a high-power Ti:sapphire laser are used, and single-shot recordings of the scattered light are made with a fast streak camera. The method is applied to the study of light scattering in paper, and a 1-ps resolution is demonstrated. The dependence of the light scattering on the basis of weight and density of the paper has been studied. A white-light continuum generated from the high-power pulses by the use of self phase modulation in water is used to study the wavelength dependence of the scattering process. A model for the propagation of light in paper has been developed and used in Monte Carlo simulations. The experimental results are used for testing this model, and absorption and scattering parameters are determined from that comparison.

Detection of short-lived intermediates in electrochemical reactions using time-resolved surface-enhanced Raman spectroscopy

p-Nitrobenzoic acid (PNBA) is studied by real-time detection of SERS spectra during time-resolved optical multichannel recording following the application of a double potential step to a Ag electrode. The spectral bands of three stable intermediate products, p-nitrosobenzoate, hydroxylamine, and azoxy compounds are observed. In addition, the transient bands of an unstable intermediate are seen at 996, 1233, and 1580 cm{sup {minus}1} with a lifetime of about 70 ms during the oxidation process of the hydroxylamine compound which itself is generated electrochemically by a 200-ms potential pulse. We suggest these bands represent the p-nitrosobenzoate free-radical anion intermediate formed during the oxidation of the hydroxylamine compound.

Time-resolved recording of the fast rise of the R1-component of the ?early receptor potential? (=ERP)

Time-resolved recording of the fast rise of the R1-component of the ?early receptor potential? (=ERP)

Time-resolved infrared spectral photography

A new technique for time-resolved (5-nsec) recording of broadband infrared absorption spectra is demonstrated. Resonantly enhanced, third-order nonlinearities of metal vapors are used both to produce a pulsed infrared continuum that interrogates the sample and to upconvert this infrared continuum into the visible. Single-shot absorption spectra of CO(2) and H(2)O bands in the region 2.55-2.85 microm were obtained.

Lifetime measurements of Rydberg states in the 6snd 1 D 2-series of BaI

We have measured the lifetimes of 10 Rydberg states from 6s8d1D2 to 6s 17d1D2 in the Ba I spectrum by time-resolved recording of the exponential decay of the resonance fluorescence in an atomic beam. The excitation was performed by use of two pulsed dye lasers simultaneously pumped by one nitrogen laser. The lifetime results differ strongly from Coulomb approximation calculations and show deviations from a(n*)3-dependence.

Increased Photographic Sensitivity for Time-Resolved Mass Spectrometer Data Recording

A tenfold increase in photographic sensitivity has been achieved for time-resolved mass spectrometer data recording with an oscillographic high speed drum camera. This has been accomplished by an optimized selection of CRT phosphor, photographic film, electron beam accelerating potential, lens aperture, sweep rate, and film processing.

The impulse breakdown voltage and time-lag characteristics of long gaps in air II. The negative discharge

Oscillographic measurement and time-resolved photographic recording have been used to examine the breakdown of rod/rod, rod/sphere and rod/plane gaps with long-duration negative impulse potentials of up to 1 MV crest. As in the positive discharge, the breakdown process is found often to cease for considerable periods on the impulse wave tail. Breakdown then occurs after a long time lag for gaps composed of rod cathodes and rod or small sphere anodes. Where the anode is large, no long time lags are observed. Measurement of the variation of breakdown strength with time during the impulse duration has been made by the superimposition of a second impulse upon the first, following a controllable delay. The results show a minimum probability of breakdown initiation after 15 μs of the applied impulse, and a subsequent slow recovery. It is suggested that this confirms the effect of the initial corona phase in causing space charge fouling of the gap. The breakdown voltage of rod/rod gaps is shown to decrease by up to 30 % when the impulse wave front is reduced from 0.50 to 0.06 μs. The breakdown strength with negative impulses is thus less than with positive impulses for wave fronts faster than 0.20 μs. For the wave form 0.50/2000 μs, the breakdown voltage is found to depend more critically upon the dimensions of the earthed electrode than is the case with positive impulses. The breakdown voltage for a rod/25 cm sphere gap is 73 % greater than for a rod/rod gap, yet also 15 % greater than for a rod/plane gap. For gap lengths between 25 and 65 cm, the breakdown voltage of the rod/plane gap is found to increase with decreasing wave front duration. The variation of the negative impulse corona with rate of potential rise and with crest voltage is examined photographically. Many of the observed variations of breakdown voltage and time-lag characteristics can be accounted for in terms of the impulse corona phase. The time-resolved photography of the negative discharge over a wide range of conditions suggests that in the range of gap lengths investigated the formation of the positive leader rather than the negative leader is the necessary forerunner of sparkover.