Time-correlated Single Photon Counting Technique Research Articles

An intriguing signal-off responsive photoelectrochemical aptasensor for ultrasensitive detection of microcystin-LR and its mechanism study

A direct formatted photoelectrochemical (PEC) aptasensing platform was constructed for ultrasensitive microcystin-LR (MC-LR) detection with AgI-nitrogen-doped graphene (AgI-NG) composites as photocathode and aptamer as recognition element. The photocurrent of the AgI-NG composites was about 10.9 times higher than that of AgI. Furthermore, to investigate the sensing mechanism, the experiments with photoluminescence (PL) and time-correlated single-photon counting (TCSPC) technique were implemented as supporting evidence. The developed aptasensor can be used for MC-LR detection in fish samples. The experimental and theoretical efforts elucidate that the theory of flow directions of electron give reasonable explanation for the sensing process: prevailing electron-hole recombination constitutes a signal-off strategy, while electron transfer playing the leading role presents a signal-on performance. Impressively, we discovered that the sensing mechanism differed significantly when applied to the same analyte despite the commonality among the detecting configuration themselves, which is dependent by the interaction between the analyte MC-LR and transducer. For the detection process, in the presence of MC-LR, the specific recognition of the targets with the sensing interface would trigger the formation of the aptamer/MC-LR complex, resulting in reduced electron transfer rate, increased charge recombination, and hence quenched photocurrent intensity of the AgI-NG with detection limits of 0.017 pM. This work will help to complement the vital theoretical explanation about the underlying sensing mechanism, and future designing of PEC aptasensors.

Recent Advances in Fluorescence Lifetime Analytical Microsystems: Contact Optics and CMOS Time-Resolved Electronics.

Fluorescence spectroscopy has become a prominent research tool with wide applications in medical diagnostics and bio-imaging. However, the realization of combined high-performance, portable, and low-cost spectroscopic sensors still remains a challenge, which has limited the technique to the laboratories. A fluorescence lifetime measurement seeks to obtain the characteristic lifetime from the fluorescence decay profile. Time-correlated single photon counting (TCSPC) and time-gated techniques are two key variations of time-resolved measurements. However, commercial time-resolved analysis systems typically contain complex optics and discrete electronic components, which lead to bulkiness and a high cost. These two limitations can be significantly mitigated using contact sensing and complementary metal-oxide-semiconductor (CMOS) implementation. Contact sensing simplifies the optics, whereas CMOS technology enables on-chip, arrayed detection and signal processing, significantly reducing size and power consumption. This paper examines recent advances in contact sensing and CMOS time-resolved circuits for the realization of fully integrated fluorescence lifetime measurement microsystems. The high level of performance from recently reported prototypes suggests that the CMOS-based contact sensing microsystems are emerging as sound technologies for application-specific, low-cost, and portable time-resolved diagnostic devices.

Spectral and kinetic features of nonradiative energy transfer in hybrid associates of colloidal quantum dots and organic dyes

The features of nonradiative resonance energy transfer in hybrid associates of colloidal CdS quantum dots (QDs) with molecules of a thiazine dye and J-aggregates of a carbocyanine dye are discussed. The case of an inhomogeneously broadened recombination band of donor (CdS QDs) luminescence is considered. We detected spectral-selective quenching of recombination luminescence of colloidal CdS QDs in association with organic dyes. The maximum of quenching occurs in the region of absorption of the acceptor (organic dye molecule). It is shown using the time-correlated single photon counting technique that the lifetime of the QD luminescence decreases synchronously with the quenching of the luminescence, but only at the wavelengths falling in the region of absorption of the dye. Based on the concept of a donor–acceptor nature of emission of CdS QDs, the dependence of the efficiency of nonradiative energy transfer on the luminescence wavelength is analyzed taking into account the Poisson distribution of its quenchers.

Fluorescence Lifetime and UV-Vis Spectroscopy to Evaluate the Interactions Between Quercetin and Its Yeast Microcapsule.

Quercetin is a fragile bioactive compound. Several works have tried to preserve it by encapsulation but the form of encapsulation (mono- or supra-molecular structure, tautomeric form), though important for stability and bioavailability, remains unknown. The present work aims at developing a fluorescence lifetime technique to evaluate the structure of quercetin during encapsulation in a vector capsule that has already proven efficiency, yeast cells. Molecular stabilization was observed during a 4-month storage period. The time-correlated single-photon counting (TCSPC) technique was used to evaluate the interaction between quercetin molecules and the yeast capsule. The various tautomeric forms, as identified by UV-Vis spectroscopy, result in various lifetimes in TCSPC, although they varied also with the buffer environment. Quercetin in buffer exhibited a three-to-four longer long-time after 24 h (changing from 6-7 to 18-23 ns), suggesting an aggregation of molecules. In yeast microcapsules, the long-time population exhibited a longer lifetime (around 27 ns) from the beginning and concerned about 20% of molecules compared to dispersed quercetin. This shows that lifetime analysis can show the monomolecular instability of quercetin in buffer and the presence of interactions between quercetin molecules and their microcapsules.

Facile fabrication of a gadolinium-doped liquid scintillator with the mixed solvent

A gadolinium-doped liquid scintillator with the mixed solvent as the matrix was prepared for a good comprehensive performance. In the ternary liquid scintillator, the β-diketonate was used as the complexing ligand of gadolinium (Gd), the combination of 20vol% pseudocumene (PC) and 80vol% linear-alkyl benzene (LAB) was chosen as the mixed solvent, and 2,5-diphenyloxazole (PPO) and 1,4-bis(2-Methylstyryl) benzene (bis-MSB) were the primary fluor and wavelength shifter, respectively. The characterizations based on the optimal formulation were conducted. The light yield was characterized by adopting the home-made optical platform device. The decay time was tested by adopting the time-correlated single photon counting (TCSPC) technique featured in high dynamic range of several orders of magnitude in light intensity. The experimental test results showed that the ternary liquid scintillator had a fairly good comprehensive performance.

Charge and Energy Transfer Dynamics in Dimethylsilylene-Spaced Aminostyrene Stilbene Monomer Using Time-Resolved Techniques.

We used transient absorption and time-correlated single photon counting (TCSPC) techniques to investigate the charge transfer reaction in monosilylene-spaced aminostyrene stilbene monomer. With 266 nm excitation, both stilbene (sti) and aminostyrene (ast) moieties were excited. In nonpolar solvents, the transient absorption band centered at 600 nm appeared promptly and is assigned to the excited state of sti*; this state relaxes at time constant 1.2-1.4 ps and is explained to proceed energy transfer to ast 1ππ*. The second transient band at 460 nm is assigned to absorption of ast 2ππ*; this state accessed from direct excitation has a lifetime 65 ps. This agrees with the observation of 85-89 ps emission decay from the TCSPC measurements. In polar solvent, an excited absorption band centered at 530 nm appeared with a rise time constant 0.2-0.6 ps. This band is assigned to the charge transfer state. This charge transfer process occurs as the acceptor fluorophore (sti) is excited and the electron moves from the occupied π orbital of donor ast to sti* forming ast+sti-. This rise time corresponds to the combined processes of charge and energy transfers. The second rise in this charge-transfer state at time constant 0.74-1.5 ps is observed and assigned to occur from electron hopping from ast 2π* orbital to sti π*. The third time constant 18-31 ps is observed and is attributed to conversion of anti to syn form in the charge-transfer state because the syn form is more polar and further stabilized in polar environment. A rapid charge transfer process in monosilylene-spaced system although two Si-C single bonds are used as spacer is possibly because of the short distance of the ast and the sti conjugated systems, resulting in π orbital overlap between donor and acceptor.

Estimating fluorescence lifetimes using extended Kalman filter

The extended Kalman filter (EKF) has been widely used in communication, signal processing and navigation control. In this Letter, the authors applied EKF, for the first time to their knowledge, to simultaneously estimate fluorescence lifetimes and instrument response functions (IRF) for time-domain fluorescence lifetime imaging microscopy (FLIM) systems (we focus on gating and time-correlated single-photon counting techniques in this work). Monte-Carlo simulations were performed to test its performances in comparison with previously reported methods. Simulation results show that the proposed algorithm can achieve comparable or better results than the others. More importantly, with EKF there is no need to measure the IRF of the FLIM system.

Analysis of Fluorescence Quenching for Newly Synthesized Biologically Active 3(2H)-pyridazinone Derivative by Aniline.

Herein, we have studied the analysis of fluorescence quenching for newly synthesized biologically active 3(2H)-pyridazinone derivative 5-(5-bromo-2-hydroxy-phenyl)-2-phenyl-2H-pyridazin-3-one [BHP] by various concentrations of aniline using UV-Visible spectroscopy, fluorescence spectroscopy and time-correlated single photon counting technique in five different solvents namely, methanol, ethanol, propan-2-ol, dimethylsulfoxide and ethyl acetate at room temperature. The fluorescence intensity of BHP molecule decrease with increasing in the aniline concentration and it is studied using the Stern-Volmer relation. The obtained Stern-Volmer plots were found to be linear in all the five solvents. The various parameters responsible for the fluorescence quenching such as quenching rate parameters (k q ), diffusion rate parameter (k d ) and the probability of quenching per encounter (p) were experimentally calculated in all five solvents. An activation energy of quenching (E a ) was calculated using the values of activation energy of diffusion (E d ) and p. It was found that the values of E a are greater than E d in all five solvents studied. Further, it is inferred that the fluorescence quenching reactions in BHP molecule are more significantly affected by activation energy processes.

Fluorescence Dynamics of a FRET Probe Designed for Crowding Studies.

Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensitive to the energy transfer from the donor to the acceptor. The results also suggest that the linker, -(GSG)6A(EAAAK)6A(GSG)6A(EAAAK)6A(GSG)6-, is rather flexible, and the observed rotational dynamics is likely to be due to a segmental mobility of the FRET pairs rather than an overall tumbling motion of a rigid probe. Comparative studies on a new construct of a FRET probe with a shorter, more flexible linker, mCerulean-(GSG)18-mCitrine, reveal enhanced energy transfer efficiency. On the millisecond time scale, fluorescence fluctuation analyses of the acceptor (excited at 488 nm) provide a means to examine the translational diffusion coefficient of the FRET probe. The results also suggest that the linker is flexible in this FRET probe, and the observed diffusion coefficient is faster than predicted as compared to the cleaved FRET probe. Our results serve as a point of reference for this FRET probe in a buffer toward its full potential as a sensor for macromolecular crowding in living cells and tissues.

Single-photon three-dimensional imaging at up to 10 kilometers range.

Depth and intensity profiling of targets at a range of up to 10 km is demonstrated using time-of-flight time-correlated single-photon counting technique. The system comprised a pulsed laser source at 1550 nm wavelength, a monostatic scanning transceiver and a single-element InGaAs/InP single-photon avalanche diode (SPAD) detector. High-resolution three-dimensional images of various targets acquired over ranges between 800 metres and 10.5 km demonstrate long-range depth and intensity profiling, feature extraction and the potential for target recognition. Using a total variation restoration optimization algorithm, the acquisition time necessary for each pixel could be reduced by at least a factor of ten compared to a pixel-wise image processing approach. Kilometer range depth profiles are reconstructed with average signal returns of less than one photon per pixel.

Time-Gated Imaging on Live Cancer Cells Using Silicon Quantum Dot Nanoparticles with Long-Lived Fluorescence

In this work we demonstrate time-gated confocal fluorescence imaging on live cancer cells immunostained by antibody-conjugated silicon quantum dot nanoparticles (SiQD-NPs) and organic dyes, for simultaneous detection of two biological targets and removal of background autofluorescence. With almost all radiative recombinations occurring through oxide-related defect states located on the SiQD surface, the SiQD-NPs have very long photoluminescence lifetimes of about 25 μs, in contrast to the nanosecond-range lifetimes of other commonly used biological fluorophores. This drastic lifetime difference enables a time-gated imaging method here, in which the time-resolved photon distribution of each pixel of a fluorescence image is measured by using a time-correlated single-photon counting technique. Then, by integrating the photon histogram of each pixel over respective time windows, the long-lived component of the fluorescence image comprising only the fluorescence emitted from the SiQD-NPs is separated from all ...

Spectroscopic interactions of titanium dioxide nanoparticles with pharmacologically active 3(2H)-pyridazinone derivative

Herein, we report the spectroscopic interactions of titanium dioxide (TiO2) nanoparticles (NPs) with pharmacologically active 3(2H)-pyridazinone derivative, viz., 5-(2-Hydroxy-4-methyl-phenyl)-2-phenyl-2H-pyridazin-3-one [HMP] in an ethanol solvent using UV–Visible spectrophotometer, fluorescence spectrophotometer and time-correlated single photon counting technique at room temperature. The observed values of absorption, fluorescence intensity and fluorescence lifetime of HMP molecule decrease with increasing in the TiO2 NPs concentration. From the linear Stern-Volmer (S-V) plot in steady state and transient state which indicates the presence of dynamic quenching. The association constant (ka) and quenching constant (KSV) have been estimated using Benesi–Hildebrand and S-V equations respectively. Further, from the fluorescence data we calculated the binding constant and number of binding sites, the results reveals that there exists one binding site in HMP molecule for TiO2 NPs. In addition, we studied the energy transfer in fluorescence quenching by Forster's non-radiative energy transfer (FRET) theory. Results, signified that the fluorescence quenching of the HMP molecule is due to the energy transfer from HMP molecule to TiO2 NPs. The present investigation may be adopted in solar energy materials and also exploited in a variety of applications such as biological sensing, medical diagnosis etc.

Ag Doping of Organometal Lead Halide Perovskites: Morphology Modification and p-Type Character

We report a simple synthetic approach to grow uniform CH3NH3PbI3 perovskite (PSK) layers free of pinholes via varied portions of silver iodide (AgI) added to the precursor solution. XRD/EDS elemental mapping experiments demonstrated nearly uniform Ag distribution inside the perovskite film. When the 1% AgI-assisted perovskite films were fabricated into a p-i-n planar device, the photovoltaic performance was enhanced by ∼30% (PCE increased from 9.5% to 12.0%) relative to the standard cell without added AgI. Measurement of electronic properties using a hall setup indicated that perovskite films show p-type character after Ag doping, whereas the film is n-type without Ag. Transients of photoluminescence of perovskite films with and without AgI additive deposited on glass, p-type (PEDOT:PSS), and n-type (TiO2) contact layers were recorded with a time-correlated single-photon counting (TCSPC) technique. The TCSPC results indicate that addition of AgI inside perovskite in contact with PEDOT:PSS accelerated the ...

Wide Scale Investigation of Protein Interactions by Automation of Fluorescent Polarization and Fluctuation Analysis

Monitoring changes in molecular conformation is essential for studying protein interactions within and between complexes. Methods such as FRET or FCS reveal limited aspects of these changes, which in many cases is insufficient for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET and FCS was developed to address this problem. In FPFA changes in protein complex mass and/or shape, the number of fluorescent subunits per complex, as well as subunit proximity (1 – 10 nm), is simultaneously detected. This multimodal approach was validated with series of 6 fluorescent oligomers composed of between 1 and 6 concatenated Venus molecules, and successfully employed to investigate structural dynamics of calcium/calmodulin-dependent protein kinase II (CaMKII) – a multimeric protein kinase that is enriched in synaptic spines and dendrites that is involved in memory and synaptic modulation, as well as microtubule-associated protein 1A/1B-light chain 3 (LC3) – a soluble protein that is a key component of autophagy, a homeostasis process responsible for the turnover of cellular components. Here we have extended FPFA method by developing a robotic microscope that can measure up to 96 samples automatically. This FPFA automation enables wide-scale biophysical analysis of protein-protein interactions, and hence facilitates the identification of drugs that target protein complexes as potential therapeutic sites for diseases of aggregation and abnormal protein-protein interactions.

Preparation and performance study of a novel liquid scintillator with mixed solvent as the matrix

A novel liquid scintillator with the mixed solvent as the matrix was prepared for obtaining a good comprehensive performance. In this ternary liquid scintillator, the combination of 20% pseudocumene (PC) and 80% linear-alkyl benzene (LAB) by volume was chosen as the mixed solvent, and 2,5-diphenyloxazole (PPO) and 1,4-bis(2-Methylstyryl) benzene (bis-MSB) were as the primary fluor and wavelength shifter, respectively. The optimum prescription was obtained with regard to the light yield. Some characterizations based on the optimal formulation were conducted. The fluorescence emission spectra and wavelength-dependent optical attenuation length of the sample were measured by the fluorescence spectrophotometer and an UV–Vis spectrometer, respectively. The light yield was characterized by adopting the home-made optical platform device. The decay time was tested by adopting the time-correlated single photon counting (TCSPC) technique featured in high dynamic range of several orders of magnitude in light intensity. The experimental test results showed that the sample had a fairly good comprehensive performance.

Adaptive Target Profile Acquiring Method for Photon Counting 3-D Imaging Lidar

A direct-detection 3-D imaging lidar is capable of acquiring a depth image of noncooperative targets in long distance, using Geiger mode avalanche photodiodes and the technique of time-correlated single-photon counting. However, conventional 3-D imaging lidar has a long data acquisition time. This paper introduces a spatially adaptive method to obtain target profile rapidly for 3-D imaging lidar by exploiting discontinuities between targets and background in the depth domain. The idea behind our strategy is using an adaptive scanning step to localize the regions near the depth boundaries and perform fine scans only to these regions. Fine scans only for those specific regions ensure the recovery accuracy while consuming much less data acquisition time, compared to a conventional high-resolution scanning lidar. Experimental results demonstrate that for the experimental scene, the data acquisition time decreases 85% by using the proposed method, without generating much distortion of the target profile. This method may be of considerable value to fields that need fast 3-D imaging, such as remote sensing and military reconnaissance.

Study of genetic evolution of oil inclusion and density of surface oil by measurement of fluorescence lifetime of crude oil and oil inclusion

By using fluorescence lifetime image microscope (FLIM) and time-correlated single photon counting (TCSPC) technique, we measured fluorescence lifetime of crude oils with density of 0.9521–0.7606 g/cm3 and multiple petroleum inclusions from Tazhong uplift of Tarim Basin. As indicated by the test results, crude oil density is closely correlated with average fluorescence lifetime following the regression equation Y=–0.0319X+0.9411, which can thus be used to calculate density of oil inclusions in relation to fluorescence lifetime and density of corresponding surface crude. For type A oil inclusions showing brown-yellow fluorescence from Tazhong 1 well in Tarim Basin, their average fluorescence lifetime was found to be 2.144–2.765 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.852–0.873 g/cm3, indicating that they are matured oil inclusions trapped at earlier stage of oil formation. For type B oil inclusions with light yellow-white fluorescence, their average fluorescence lifetime was found to be 4.029–4.919 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.784–0.812 g/cm3, indicating that they are higher matured oil inclusions trapped at the second stage of oil formation. For type C oil inclusions showing light blue-green fluorescence, their average fluorescence lifetime was found to be 5.063–6.168 ns, so the density of surface crude corresponding to crude trapping these oil inclusions is 0.743–0.779 g/cm3, indicating that they are highly-matured light oil inclusions trapped at the third stage of oil formation.

Optical and scintillation characteristics of Gd2YAl2Ga3O12:Ce and Lu2YAl2Ga3O12:Ce single crystals

The optical and scintillation characteristics of Gd2YAl2Ga3O12:Ce and Lu2YAl2Ga3O12:Ce single crystals are investigated. At 662keV γ-rays, light yield (LY) of 37,900ph/MeV and energy resolution of 7.0% obtained for Gd2YAl2Ga3O12:Ce are superior to those of 18,900ph/MeV and 11.5% obtained for Lu2YAl2Ga3O12:Ce. Scintillation decays are measured using the time-correlated single photon counting technique. A fast component decay time of 45ns with relative intensity of 88% obtained for Lu2YAl2Ga3O12:Ce is superior to that of 50ns (65%) for Gd2YAl2Ga3O12:Ce. The linear attenuation coefficient at 662keV γ-rays is also determined and discussed.

On the origin of multiexponential fluorescence decays from 2-aminopurine-labeled dinucleotides.

The fluorescent probe 2-aminopurine (2Ap) has been used for decades to study local conformational fluctuations in DNA. Steady-state and time-resolved measurements of 2Ap fluorescence have been used to predict specific conformational states through suitable modeling of the quenching of the fluorescence of a 2Ap residue incorporated site-specifically into a DNA strand. The success of this approach has been limited by a lack of understanding of the precise factors responsible for the complex, multiexponential decays observed experimentally. In this study, dinucleotides composed of 2Ap and adenine were studied by the time-correlated single-photon counting technique to investigate the causes of heterogeneous emission kinetics. Contrary to previous reports, we argue that emission from 2Ap that is stacked with a neighboring base contributes negligibly to the emission signals recorded more than 50 ps after excitation, which are instead dominated by emission from unstacked 2Ap. We find that the decay kinetics can be modeled using a continuous lifetime distribution, which arises from the inherent distance dependence of electron transfer rates without the need to postulate a small number of discrete states with decay times derived from multiexponential fits. These results offer a new perspective on the quenching of 2Ap fluorescence and expand the information that can be obtained from experiments.

Subnanosecond Emission Dynamics of AT DNA Oligonucleotides.

UV radiation creates excited electronic states in DNA that can decay to mutagenic photoproducts. When excited states return to the electronic ground state, photochemical injury is avoided. Understanding of the available relaxation pathways has advanced rapidly during the past decade, but there has been persistent uncertainty, and even controversy, over how to compare results from transient absorption and time-resolved emission experiments. Here, emission from single- and double-stranded AT DNA compounds excited at 265 nm was studied in aqueous solution using the time-correlated single photon counting technique. There is quantitative agreement between the emission lifetimes ranging from 50 to 200 ps and ones measured in transient absorption experiments, demonstrating that both techniques probe the same excited states. The results indicate that excitations with lifetimes of more than a few picoseconds are weakly emissive excimer and charge transfer states. Only a minute fraction of excitations persist beyond 1 ns in AT DNA strands at room temperature.