Time-correlated Single Photon Counting Technique Research Articles

Study of conformational transitions of i-motif DNA using time-resolved fluorescence and multivariate analysis methods.

Recently, the presence of i-motif structures at C-rich sequences in human cells and their regulatory functions have been demonstrated. Despite numerous steady-state studies on i-motif at neutral and slightly acidic pH, the number and nature of conformation of this biological structure are still controversial. In this work, the fluorescence lifetime of labelled molecular beacon i-motif-forming DNA sequences at different pH values is studied. The influence of the nature of bases at the lateral loops and the presence of a Watson–Crick-stabilized hairpin are studied by means of time-correlated single-photon counting technique. This allows characterizing the existence of several conformers for which the fluorophore has lifetimes ranging from picosecond to nanosecond. The information on the existence of different i-motif structures at different pH values has been obtained by the combination of classical global decay fitting of fluorescence traces, which provides lifetimes associated with the events defined by the decay of each sequence and multivariate analysis, such as principal component analysis or multivariate curve resolution based on alternating least squares. Multivariate analysis, which is seldom used for this kind of data, was crucial to explore similarities and differences of behaviour amongst the different DNA sequences and to model the presence and identity of the conformations involved in the pH range of interest. The results point that, for i-motif, the intrachain contact formation and its dissociation show lifetimes ten times faster than for the open form of DNA sequences. They also highlight that the presence of more than one i-motif species for certain DNA sequences according to the length of the sequence and the composition of the bases in the lateral loop.

Free and bound Thioflavin T molecules with ultrafast relaxation: implications for assessment of protein binding and aggregation

Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ~1 to ~1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity’s dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ~100 ps instrument response function. This results in the situation when a small fraction (~1%) of ThT molecules with a relatively slow decay (τ ~ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ~ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay with a fluorescence lifetime determined by the TCSPC as an indicator.

Excited state intramolecular single proton transfer mechanism of pigment yellow 101 in solid state: Experiment and DFT calculation.

To investigate fluorescence mechanism of Pigment Yellow 101 (P. Y. 101) in solid state, three aromatic aldehyde azines (1-3) including P. Y. 101 have been synthesized and compared with each other. Results indicated that P. Y. 101 prepared by solvothermal method is actually the mixture of two polymorphs, whose molecular packing mode can be transformed into each other by recrystallizing or external stimuli such as pressure and grinding. The ESIPT properties of 1-3 were investigated by DFT/TD-DFT calculations and time-correlated single photon counting (TCSPC) technique. Both experimental and theoretical results revealed that the dual fluorescence properties of P. Y. 101 in solid state are ascribed to the excited-state intramolecular single proton transfer fluorescence emissions of two structurally different polymorphs rather than the results of the sequential or concerted excited-state intramolecular double proton transfers, which provide a potential valuable tool for developing multistimuli-responsive luminescent materials.

Measurements of cesium mixing and quenching cross sections in methane gas: understanding sources of heating in cesium vapor lasers.

Accurate modeling of the operation of diode-pumped alkali lasers is a critical step toward the design of high-powered devices. We present precision measurements for the Cs-CH4 62P3/2 → 62P1/2 mixing cross section and the 62P3/2,1/2 → 62S1/2 quenching cross section, which are important parameters in understanding the operation and, in particular, the heat generated in a cesium vapor laser. Measurements are carried out using ultrafast laser pulse excitation and observation of fluorescence due to collisional excitation transfer in time is done using the technique of time-correlated single-photon counting. Mixing rate measurements are acquired over methane pressures of 10 - 40 Torr, resulting in a Cs-CH4 62P3/2 → 62P1/2 mixing cross section of (1.40 ± 0.08) × 10-15 cm2, while quenching rate measurements are carried out over methane pressures of 500 - 4000 Torr, resulting in a 62P3/2,1/2 → 62S1/2 quenching cross section of (1.57 ± 0.03) × 10-18 cm2. These results suggest only a slight contribution to the heating of a cesium vapor laser is due to Cs 62P quenching, contrary to previous studies. We also discuss additional possible sources of energy transfer from upper excited states of Cs.

About the Development and Dynamics of Microdischarges in Toluene-Containing Air

The development of microdischarges and the inception dynamics of subsequent microdischarges in an electrode arrangement consisting of a metal pin and a hemispherical dielectric-covered electrode, operated in air with a small toluene admixture, is studied. The discharge is operated with sinusoidal high voltage. A gated ICCD camera and a current probe enable the recording of images and current pulses of the single microdischarges, respectively, while the spatio-temporally resolved development is measured with a multi-dimensional time-correlated single photon counting technique. The overall discharge dynamics changes significantly if a concentration of 35 ppm toluene is added to dry air. A lower high voltage amplitude than in dry air is needed for stable discharge operation. This can be explained by the lower ionization energy of toluene compared to molecular oxygen and nitrogen. The microdischarge development is the same with or without admixture, i.e. a positive (cathode directed) streamer mechanism is observed. Lower mean power is dissipated into the discharge when toluene is admixed. The main effect caused by toluene admixture is the suppression of high-energy microdischarges in case of the cathodic pin half-cycle of the sinusoidal high voltage. The influence on the inception voltage by additional ionization mechanisms and volume memory effects, the consumption of energetic electrons for toluene decomposition reactions, and the modification of the surface by plasma treatment are discussed as possible reasons.

Solvent-Assisted Enhanced Emission of Cationic Perylene Diimide Supramolecular Assembly in Water: A Perspective from Experiment and Simulation

We report a solvent-assisted increase in the emission of a cationic perylene diimide derivative, which is water-soluble in nature. Favorably, all the emission-assisting solvents used in all the experiments are water-miscible such as ethanol, tetrahydrofuran (THF), or dimethyl sulfoxide (DMSO). The preliminary assumption of associated fluorescent assembly of perylene stacks (via spectroscopic analysis) has been further explored with dynamic light scattering (DLS), nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and time-correlated single photon counting (TCSPC) techniques. Spectroscopically observed drastic enhancement in the emission intensity seems to be born out of a major change in the assembly pattern of the interacting hydrophobic surfaces of perylene derivatives. These changes further have a macroscopic impact on the DLS or powder XRD outcomes. Furthermore, molecular dynamics simulations have been performed to interpret the nature of aggregation of perylene oligomers in water and in an et...

NIR-II fluorescence in vivo confocal microscopy with aggregation-induced emission dots

Significantly reduced tissue scattering of fluorescence signals in the second near-infrared (NIR-II, 1,000–1,700 nm) spectral region offers opportunities for large-depth in vivo bioimaging. Nowadays, most reported works concerning NIR-II fluorescence in vivo bioimaging are realized by wide-field illumination and 2D-arrayed detection (e.g., via InGaAs camera), which has high temporal resolution but limited spatial resolution due to out-of-focus signals. Combining NIR-II fluorescence imaging with confocal microscopy is a good approach to achieve high-spatial resolution visualization of biosamples even at deep tissues. In this presented work, a NIR-II fluorescence confocal microscopic system was setup. By using a kind of aggregation-induced emission (AIE) dots as NIR-II fluorescent probes, 800 μm-deep 3D in vivo cerebrovascular imaging of a mouse was obtained, and the spatial resolution at 700 μm depth could reach 8.78 μm. Moreover, the time-correlated single photon counting (TCSPC) technique and femtosecond laser excitation were introduced into NIR-II fluorescence confocal microscopy, and in vivo confocal NIR-II fluorescence lifetime microscopic imaging (FLIM) of mouse cerebral vasculature was successfully realized.

Optical properties of composited TiO2-aluminium-doped ZnS photoanode

Abstract A composited photoanode for optoelectronic devices have widely attracted much attention for many decades. In this work, wide band gap semiconductors, ZnS, and aluminium-doped ZnS were synthesized through simply chemical synthetic route with band gap energy of 3.67 – 3.69 eV. The thin films of TiO2/ZnS and TiO2/aluminium-doped ZnS composites with various concentration were prepared by doctor blade technique. By means of X-ray diffraction, the thin film structures of TiO2 is rarely dominated by ZnS or aluminium-doped ZnS. A ruthenium sensitizer (N719) was sensitized on the composited thin films. The absorption and photoluminescence of the composited photoanode were characterized, indicating the band gap energy of 3.41 eV for all thin films. It was observed that both ZnS and aluminium-doped Zns reduce the amount of dye uptake. Also, the photoluminescence of dye sensitized TiO2/ZnS and TiO2/aluminium-doped ZnS photoanode was rather blue-shifted and enhanced with the increasing of ZnS or aluminium-doped ZnS amount. Moreover, using Time Correlated Single Photon Counting technique (TCSPC), the emission lifetimes of dye sensitized TiO2, TiO2/ZnS and TiO2/aluminium-doped ZnS anodes are 3.32 ns, 8.40 ns and 6.42 ns, respectively which indicate the charge injection from excited dye to photoanode.

Multiparametric Time-Correlated Single Photon Counting Luminescence Microscopy.

Classic time-correlated single photon counting (TCSPC) technique involves detection of single photons of a periodic optical signal, registration of the photon arrival time in respect to the reference pulse, and construction of photon distribution with regard to the detection times. This technique achieves extremely high time resolution and near-ideal detection efficiency. Modern TCSPC is multi-dimensional, i.e., in addition to the photon arrival time relative to the excitation pulse, spatial coordinates within the image area, wavelength, time from the start of the experiment, and many other parameters are determined for each photon. Hence, the multi-dimensional TCSPC allows generation of photon distributions over these parameters. This review describes both classic and multi-dimensional types of TCSPC microscopy and their application for fluorescence lifetime imaging in different areas of biological studies.

Hyperspectral wide-field time domain single-pixel diffuse optical tomography platform.

We present the design and comprehensive instrumental characterization of a time domain diffuse optical tomography (TD-DOT) platform based on wide-field illumination and wide-field hyperspectral time-resolved single-pixel detection for functional and molecular imaging in turbid media. The proposed platform combines two digital micro-mirror devices (DMDs) to generate structured light and a spectrally resolved multi-anode photomultiplier tube (PMT) detector in time domain for hyperspectral data acquisition over 16 wavelength channels based on the time-correlated single-photon counting (TCSPC) technique. The design of the proposed platform is described in detail and its characteristics in spatial, temporal and spectral dimensions are calibrated and presented. The performance of the system is further validated through a phantom study where two absorbers in glass tubes with spectral contrast are mapped in a turbid medium of ~20 mm thickness. The method presented here offers the potential of accelerating the imaging process and improving reconstruction results in TD-DOT and thus facilitates its wide spread use in preclinical and clinical in vivo imaging scenarios.

Synthesis, photophysical and antioxidant properties of pyrrolo[3,2-c]carbazole and dipyrrolo[3,2-c:2′,3′-g]carbazole compounds

The synthesis of (6-ethyl-1,6-dihydropyrrolo[3,2-c]carbazol-2-yl)methanol 5 and (6-ethyl-6,11-dihydro-1H-dipyrrolo[3,2-c:2′,3′-g]carbazole-2,10-diyl)dimethanol 6 were achieved via the reduction of methyl pyrrolo carbazole carboxylate 3 and methyl dipyrrolo carbazole carboxylate 4, respectively. The structures of hydroxymethyl-pyrrolocarbazoles 5 and 6 were supported by FT-IR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 1H and 13C NMR spectroscopy. The photophysical properties of the targeted compounds 3–6 were investigated by employing absorption and fluorescence spectroscopy in different common organic solvents. Also, the fluorescence lifetime (τF) of the compounds was measured utilizing a time-correlated single-photon counting technique in tetrahydrofuran. Antioxidant activities of compounds 3–6 were determined by employing three different assays, namely DPPH radical scavenging, ABTS cation radical decolarization and cupric reducing antioxidant capacity. The results revealed that the ABTS cationic scavenging activity assay was found to be the most sensitive method for the determination of inhibition values.

Single microdischarges in a barrier corona arrangement with an anodic metal pin: discharge characteristics in subsequent breakdowns

Metal pin-to-dielectric-covered electrode arrangements can be considered as a combination of corona discharge and dielectric barrier discharge. In the current study, the discharge is operated in dry air at atmospheric pressure. Sinusoidal voltage is applied to the dielectric-covered hemispherical electrode, and the metal pin electrode is grounded. Using an ICCD camera and a current probe (Rogowski coil), images and current pulses of single microdischarges (MDs) are recorded simultaneously. In addition, a time-correlated single photon counting technique is used to record the spatio-temporally resolved development of the MDs. The appearance and properties of the MDs in the two polarities of the applied voltage differ significantly. In this contribution, only the results of the negative half-cycle, i.e. anodic pin, are presented. In this half-cycle, for the voltage amplitude being applied, two MDs appear in each applied voltage cycle. The first MDs leave a positive charge on the surface of the dielectric, which has considerable influence on the propagation and properties of the second MDs, namely slower propagation of the cathode directed streamer in the dielectric vicinity, further expansion of the plasma on the dielectric surface and longer presence of the bulk plasma in the gap.

High resolution photon time-tagging lidar for atmospheric point cloud generation.

The application of time-correlated single photon counting hardware and techniques to atmospheric lidar is presented. The results establish the viability of adapting photon time-tagging techniques to atmospheric lidar systems, facilitating high-range resolution (millimeter-level precision) and dynamic system observing capabilities that address the variety of atmospheric scatterers often present in atmospheric lidar profiles. The technique is demonstrated through measurements made by a high repetition rate, low pulse energy, elastic scattering, photon counting lidar. Detection probabilities with a non-zero system dead-time are derived and tested using acquired data. Atmospheric point cloud generation and the statistical implications on data retrievals utilizing this approach are presented. The results show an ability to preserve backscattered intensities while generating photon detections at picosecond resolution from a variety atmospheric scatterers.

Comparative Study of GdLu2Al2Ga3O12:Ce and GdY2Al2Ga3O12:Ce Scintillation Crystals for $\gamma$ -Ray Detection

The scintillation characteristics of Czochralski-grown GdLu2Al2Ga3O12:Ce and GdY2Al2Ga3O12:Ce single crystals were compared for $\gamma$ -ray detection. At 662-keV $\gamma$ -rays, light yield (LY) of 30700 photons/MeV obtained for GdLu2Al2Ga3O12:Ce is higher than that of 27200 photons/MeV obtained for GdY2Al2Ga3O12:Ce, while its energy resolution is inferior (14% versus 10.6%) due to a larger nonproportionality of LY. Scintillation decays were measured using the time-correlated single photon counting technique. A fast component decay time of 48 ns with relative intensity of 71% obtained for GdLu2Al2Ga3O12:Ce is superior to that of 106 ns (57%) for GdY2Al2Ga3O12:Ce. The coincidence time spectrum was measured in reference to a fast BaF2 scintillator, and coincidence time resolution was discussed.

Fast Long-Range Photon Counting Depth Imaging With Sparse Single-Photon Data

Fast depth imaging of noncooperative targets at a range of up to 900 m is demonstrated based on the time-of-flight time-correlated single-photon counting technique. Experimental results shown that our image system has the ability of reconstructing depth images with data of less than one photon per pixel. Especially, utilizing a modified total variation regularization algorithm with an optimal initial value, the acquisition time necessary for each pixel could be reduced by a factor of 8 compared to the traditional median filter approach, and the image processing time is extremely improved. This depth imaging system is a low-light-level device for a variety of applications, such as target detection, space surveillance, and distance measurement.

Dynamic time-correlated single-photon counting laser ranging

We demonstrate a photon counting laser ranging experiment with a four-channel single-photon detector (SPD). The multi-channel SPD improve the counting rate more than 4×107 cps, which makes possible for the distance measurement performed even in daylight. However, the time-correlated single-photon counting (TCSPC) technique cannot distill the signal easily while the fast moving targets are submersed in the strong background. We propose a dynamic TCSPC method for fast moving targets measurement by varying coincidence window in real time. In the experiment, we prove that targets with velocity of 5 km/s can be detected according to the method, while the echo rate is 20% with the background counts of more than 1.2×107 cps.

Fast recognition of single quantum dots from high multi-exciton emission and clustering effects.

Recognition of single quantum dots (QDs) from high multi-exciton emission and clustering effects is challenging using the conventional second-order correlation function method. Here we demonstrate a method for fast recognizing single QDs based on the probabilities of detecting single- and two-photon events. The time-tagged, time-resolved and time-correlated single-photon counting technique is applied to effectively remove multi-exciton emission and low-counting background. By this way, single QDs can be fastly recognized by the spatial coincidence-counting model. In addition, the fast recognition of single QDs by using the collected photons during the confocal scanning imaging process has been achieved synchronously.

Luminescence and scintillation characteristics of (GdxY3-x)Al2Ga3O12:Ce (x = 1,2,3) single crystals

The luminescence and scintillation characteristics of Czochralski-grown (GdxY3-x)Al2Ga3O12:Ce (x = 1,2,3) single crystals are presented. With increasing Gd content in this garnet host, the 5d2 absorption band was blue-shifted while the 5d1 absorption and 5d1 → 4f emission bands were red-shifted due to an increase in the crystal field splitting of the 5d levels. In addition, the luminescence quenching temperature of the Ce3+emission and activation energy for thermal quenching decreased with increasing Gd content. The Gd3+ → Ce3+ energy transfer was evidenced by photoluminescence excitation spectra of Ce3+ emission. At 662 keV γ - rays, the light yield (LY) of 48,600 ph/MeV and energy resolution of 6.5% was measured for a Gd3Al2Ga3O12:Ce crystal. Scintillation decay measurements were performed using the time-correlated single photon counting technique. Superior time resolution of Gd3Al2Ga3O12:Ce is due to its high LY and fast scintillation response. The total mass attenuation coefficients at 60 and 662 keV γ - rays were also determined.

Time-resolved gamma spectroscopy of single events

In this article we present a method of characterizing scintillating materials by digitization of each individual scintillation pulse followed by digital signal processing. With this technique it is possible to measure the pulse shape and the energy of an absorbed gamma photon on an event-by-event basis. In contrast to time-correlated single photon counting technique, the digital approach provides a faster measurement, an active noise suppression, and enables characterization of scintillation pulses simultaneously in two domains: time and energy. We applied this method to study the pulse shape change of a CsI(Tl) scintillator with energy of gamma excitation. We confirmed previously published results and revealed new details of the phenomenon.

Effect of sugars on the dynamics of hydrophilic fluorophores confined inside the water pool of anionic reverse micelle: A spectroscopic approach

In this article, we have investigated the structural and chemical effects of two sugar molecules, namely sucrose and sucralose on the structure of anionic reverse micelle (RM) AOT (aerosol-OT; sodium bis(2-ethylhexyl)sulfosuccinate)/n-heptane. The artificial sugar alternative sucralose shows very unique physical properties unlike other sugar molecules including sucrose and it is the reason behind choosing these two sugars in our study. The variation of shapes and sizes of the water pool of RM in presence of the two sugar molecules are investigated using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) measurements. It is observed that addition of both the sugar molecules swell the water pool of the RM and this enhancement is greater for sucralose. Moreover, we have employed time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS) techniques for hydrophilic probes Coumarin 343(C343) and Rhodamine 6G (R6G) respectively inside the RM and we have observed that these two sugar molecules strongly retard the solvation and rotational dynamics of C343 and the translational diffusion of R6G inside the RM. Replacement of the smaller sized water molecules by the larger sized sugar molecules at the interface of RM through stronger hydrogen bonding with the interfacial sulfonate moiety of AOT increases the microviscosity and this may be the probable reason behind the slow dynamics of the probes in presence of sucrose. Along with this factor, strong electrostatic interaction of highly polar molecule sucralose with RM interface and with the dye molecules further retards the dynamics of fluorophores. Therefore, sucralose molecule exhibits its distinct feature by swelling the water pool inside the RM and slowing down the dynamics of the hydrophilic probes to a greater extent compared to sucrose which provides a new aspect of RM and sugar interaction.