Single Photon Counting Technique Research Articles

Temperature-dependent Rotational Dynamics of a 3-(benzo[d]Thiazol-2-yl)-7-diethylamino)-2H-chromen-2-one non-polar Probe in n-pentanol, n-nonanol, and n-decanol Solvents.

Rotational dynamics of non-polar probe 3-(benzo[d]thiazol-2-yl)-7-(diethylamino)-2H-chromen-2-one (3BT7D2H-one) and in n-pentanol, n-nonanol, and n-decanol alcohol solvents is studied using steady-state fluorescence depolarization and time-correlated single photon counting technique with varying temperature. Experimental observations indicate that 3BT7D2H-one rotates slowly in n-decanol solvent compared to other chosen solvents. Rotational motion is analyzed using the Stoke 's-Einstein-Debye (SED) model. 3BT7D2H-one solute rotational dynamics is satisfactorily described by the SED slip boundary limit in all three solvents.

Storehouse of Peculiar Supramolecular Architecture: Probing the Charge-Transfer Phenomenon and Effect of Altering the pH in a Meta-Oriented Single Donor-Double Acceptor Fluorophore.

The investigation of excited-state intramolecular charge transfer (ESICT) has been a fascinating area of research. Although the ESICT events have been studied mostly for para-disubstituted donor-acceptor type molecules, the meta-oriented donor-acceptor type molecules have also shown tremendous potential as ESICT active molecules. In the current work, a small fluorescent probe diethyl 5-amino isophthalate (DE-5A-IPA) was investigated as a potential model to investigate ESICT events in the solution as well as solid phase. DE-5A-IPA was synthesized easily starting from commercially available 5-amino isophthalic acid in excellent yield. In the solid state, differing extents of CH···π-type binding led to formation of fully planar and puckered "cyclobutane" mimic supramolecular architecture, as evidenced from the crystal structure analysis. In addition, the single crystal structure of DE-5A-IPA shows that the donor (-NH2) and acceptor (-CO2Et) are situated in the same plane, thereby assuring the prerequisites of a through-space charge transfer. DE-5A-IPA undergoes noticeable Stokes shift (in cm-1) when the polarity of the medium was changed (4820 cm-1 in hexane; 9375 cm-1 in water). The excited-state dipole moment (μe) was calculated to be 4.0 units higher than the ground-state dipole moment (μg). DE-5A-IPA had an appreciable quantum yield (0.10 ± 0.03 to 0.27 ± 0.04). The ESICT phenomenon was also investigated by ground- and excited-state structural calculations using Gaussian 16 software. The excited-state lifetime measured by the time correlated single photon counting technique was found to vary with the polarity of the solvent, thereby providing further support to the ESICT phenomenon being operative in DE-5A-IPA. Taking advantage of the -NH2 group (which is susceptible to protonation) in DE-5A-IPA, steady state and time-resolved photodynamics were investigated in solutions of varying pH values. Interestingly, the emission quantum yields as well as the emission lifetime increase with an increase in pH value, thereby establishing DE-5A-IPA as a pH-sensitive probe. The current findings shall boost the understanding of meta-oriented ESICT enabled compounds in terms of their excited-state photophysics.

The use of low-frequency current fluctuations in measuring the mobility of holes in the MEH-PPV polymer

A diagnostic method based on the evaluation of low-frequency current fluctuation spectra is presented. When measuring the current through a p-type MEH-PPV sample, the occurrence of fluctuation is observable which can be measured with an AC amplifier. A model has been proposed that proves that the fluctuations originate from the interaction between the valence band and the band gap traps. The mean value of the amplitudes of these fluctuations increases linearly with decreasing frequency with a slope from which the product of mobility and lifetime of current carriers µpτp= (9 ± 3) × 10−15 cm2V−1 was obtained. The hole lifetime of (0.27 ± 0.01) ns was evaluated from the luminescence relaxation using the time-correlated single photon counting (TCSPC) technique. The mobility value (3 ± 1) × 10−5 cm2 V−1s−1 calculated using the above methods was compared with the mobility 1.8 × 10−5 cm2 V−1s−1 determined by the CELIV method (Charge extraction by linearly increasing voltage) and good agreement was obtained.

Quantitative analysis of transferrin uptake into living cells at single-molecule level

Endocytosis, apart from providing a mechanism for cells to take up nutrients, is the principal route of entry for many nanomedicines. The evaluation of therapeutic delivery efficiency without providing quantitative parameters, like the number of molecules entering the cell and the time of their entry, is very limited. Despite advances in single-molecule methods for in-cell quantitative measurements, they have not become widely used in the study of endocytosis. Their application, however, is challenging owing to the appropriate experimental design and applicable analysis methods. Here, by using an integrated approach based on the multidimensional time-correlated single-photon counting (TCSPC) technique, we demonstrate the quantitative method to measure the time- and concentration-dependent uptake of TRITC-transferrin into living cells with single-molecule sensitivity. The methodology opens possibilities for quantitatively assessing the delivery efficiency of fluorescent molecules into living cells.

Tracing the electron transport behavior in quantum-dot light-emitting diodes via single photon counting technique

The electron injection and transport behavior are of vital importance to the performance of quantum-dot light-emitting diodes. By simultaneously measuring the electroluminescence-photoluminescence of the quantum-dot light-emitting diodes, we identify the presence of leakage electrons which leads to the discrepancy of the electroluminescence and the photoluminescence roll-off. To trace the transport paths of the leakage electrons, a single photon counting technique is developed. This technique enables us to detect the weak photon signals and thus provides a means to visualize the electron transport paths at different voltages. The results show that, the electrons, except those recombining within the quantum-dots, leak to the hole transport layer or recombine at the hole transport layer/quantum-dot interface, thus leading to the reduction of efficiency. By reducing the amount of leakage electrons, quantum-dot light-emitting diode with an internal power conversion efficiency of over 98% can be achieved.

Near-zero distortion in TCSPC at more than one photon per excitation period: experimental validation.

The time-correlated single-photon counting (TCSPC) technique is widely renowned for its capability of reconstructing rapid and weak light signals with exceptional sensitivity and sub-picosecond timing resolution. Unfortunately, the speed of TCSPC has been historically severely limited to avoid a phenomenon known as pileup distortion. For this reason, the count rate of a classic TCSPC acquisition channel is kept below a few percent of the laser excitation rate (usually 1%-5%). In this work, we experimentally validate a novel, to our knowledge, TCSPC theory recently reported that effectively overcomes such a limitation and finally achieves high-speed operation without distortion. Exploiting a single-photon avalanche diode (SPAD), in this paper we show how to acquire additional information about the status of the system at run time, and by combining it with the classic TCSPC data histogram, we report how a count rate of approximately 60% of the excitation frequency with near-zero distortion can indeed be achieved with a commercial system.

Bismuth (Bi3+) based lead-free halide perovskitoid for light driven applications: A potential low-toxic alternative for lead (Pb2+)

Bismuth-based perovskite-like materials are recognised as feasible optoelectronic alternatives to lead-based perovskites. High toxicity and limited environmental stability of Pb-based perovskite are the principal hurdles for their practical use, whereas Bismuth (Bi3+) based materials exhibit relatively low toxicity. In this study, we fabricated (FPEA)BiI4 (FPEA = Fluorophenylethyl amine, as a hydrophobic aromatic spacer) and explored its compatibility in the field of photocatalysis and solar cells. UV–Vis, photoluminescence (PL) and time-correlated single photon counting (TCSPC) techniques were used to understand the optoelectronic properties of the material. These fabricated thin films were used towards toxic MBT remediation. Solar cells (in n-i-p configuration) were also fabricated using (FPEA)BiI4 and their photo conversion efficiency (PCE) was tested under AM1.5 illumination.

Quantifying distortion in time-correlated single photon counting: a universal parameter.

One major drawback of the classic time-correlated single photon counting (TCSPC) technique is pileup-related distortion. To keep it under a reasonable level, the maximum count rate has to be reduced, posing a serious limitation to the overall measurement speed. This means that there is an intrinsic trade-off between speed and distortion: either count rate is raised, but distortion is worsened, or distortion is minimized at the expense of speed. In both cases, it is impossible to precisely evaluate the degree of distortion introduced. Here comes our new, to the best of our knowledge, figure of merit, which is able to provide a numerical estimate of the distortion whatever the signal shape is, marking a real turning point in the way of doing TCSPC. In this article, this new parameter will be defined and its effectiveness will be demonstrated by means of mathematical computations.

Assessing the overflowing pile-up effect on the photoluminescence lifetime of nanomaterials.

The tunable complex emissive states with nanosecond to microsecond lifetimes in nanomaterials, arise due to their structural heterogeneity, enabling them with a wide range of advanced optoelectronic applications. However, understanding the complex photoluminescence lifetime in these nanomaterials is critically challenged by the overflowing pile-up effect, which occurs due to the high repetition rate of the light source in the time-correlated single photon counting (TCSPC) technique. Here, we provide a quantitative lifetime analysis, especially in metal nanoclusters, metal complexes, and semiconductor quantum dots, which suggests that the same experimental parameters can mislead the lifetime data interpretation for long-ranged luminescent nanomaterials. We demonstrate that the overflowing pile-up effect could be fatal while analyzing the excited state lifetime. Furthermore, we provide the optimized parameters that could be used to get the actual lifetime data of samples. We hope that our findings will be crucial in obtaining the error-free and accurate excited state dynamics of these long-range lifetime nanomaterials.

Two-photon excited luminescence of sulfur quantum dots for heavy metal ion detection.

Spectrally-resolved third-order nonlinear optical properties of water-dispersed sulfur quantum dots (SQDs) were investigated in the wavelength range from 740 nm to 820 nm with the two-photon excited emission technique using a tunable femtosecond laser system. The maximum value of the two-photon absorption (TPA) cross-section (σ2) for ∼5.4 nm size SQDs was found to be 185 GM (Goeppert-Mayer unit), while the two-photon brightness (σ2 × η) was found to be 1.5 GM at 780 nm, the wavelength being in the first biological transmittance window. The TPA properties are presented here as appropriate cross-sections normalized per molecular weight which enables meaningful comparison of the nonlinear factors of the studied quantum dots with those of various nanomaterials. The optimized TPA properties of these hydrophilic colloidal SQDs may be potentially useful for detection of Fe3+ metal ions. The experimentally determined limit of Fe3+ detection for both one- and two-photon regime was 10 μmol L-1 (0.6 μg mL-1). Förster resonance energy transfer between SQDs as donors and Fe3+ metal ions as acceptors was confirmed as one of the possible detection mechanisms using a time-correlated single photon counting technique.

Modulation of excimer formation and photostability of avobenzone inside the nanocavities of cyclodextrins

Avobenzone (AVZ), a UVA filter, is extensively used in sunscreen lotion and protects the skin from harmful UVA radiation. Despite their importance in sunscreen formulation, less attention has been given to its light induced excited state properties. Herein, we have explored the photophysical properties of AVZ in aqueous solutions and cyclodextrins (CDs) complexes by employing steady-state, time-resolved fluorescence, and molecular modelling tools followed by photostability analysis. AVZ exhibits dual emission from monomer and excimer forms in aqueous solutions. Spectroscopic studies revealed that AVZ forms multiple inclusion complexes with β-CD and γ-CD depending upon the host’s cavity size. These versatile host–guest complexes resulted in the modulation of the excimer formation of AVZ. The presence of α-CD and β-CD complexes with AVZ inhibited the excimer forms. In contrast, γ-CD facilitated excimer formation because its larger cavity could encapsulate the AVZ dimer. Stoichiometry and association constants for multiple host–guest complexes were determined using Benesi-Hildebrand (BH) plot. Job’s plot was used to confirm the stoichiometry of complexes. Time correlated single photon counting (TCSPC) technique was used to depict the excited state lifetime values of AVZ:CD inclusion complexes. Furthermore, these inclusion complexes were characterized by Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. Molecular docking followed by semiempirical calculations confirm the experimental results on stoichiometries and binding constants of host–guest complexes. UV light-induced absorption kinetics revealed that the photostability of AVZ inside the β-CD cavity is much better than other inclusion complexes of CDs. Overall, the present study helps us in further understanding of excited state behavior of AVZ inside hydrophobic nanocavities.

Pulse-sampling fluorescence lifetime imaging: evaluation of photon economy.

This Letter presents an experimental study comparing the photon rate and photon economy of pulse sampling fluorescence lifetime imaging (PS-FLIm) with the conventional time-correlated single photon counting (TCSPC) technique. We found that PS-FLIm has a significantly higher photon detection rate (200 MHz) compared with TCSPC (2-8 MHz) but lower photon economy (4-5 versus 1-1.3). The main factor contributing to the lower photon economy in PS-FLIm is laser pulse variability. These results demonstrate that PS-FLIm offers 25× faster imaging speed than TCSPC while maintaining room light rejection in clinical settings. This makes PS-FLIm a robust technique for clinical applications.

Photoluminescence of high energy xenon ion irradiated magnesium aluminate spinel single crystals

Laser confocal microscopy technique (60 ps laser pulse excitation at 445 nm) and a time-correlated single photon counting (TCSPC) technique have been used to study the photoluminescence (PL) in 152 MeV Xe ion irradiated MgAl2O4 single crystals. It was shown that radiation defects produced by swift xenon ions give rise to broad luminescence band positioned in the 470–800 nm wavelength range. The measurements of the PL decay curves have evidenced that lifetime of defect-related PL signal gradually increases from 6 to 10 ns in 500–620 nm spectral range and decreases with ion fluence increasing. The decrease of the luminescence intensity observed after fluences ∼1013 cm−2 is attributed to overlapping of individual ion track core regions.

Exploring novel and fast stability or sameness evaluation tool for different categories of injectable formulations

The establishment of drug product stability and sameness is the heart of generic formulation development. For regulatory filing, various instrumental methods are used on a case basis to establish the generic and innovator product sameness in multiple aspects. Here in the present study, we explored the applicability of the Time-correlated single photon counting (TCS-PC) technique as a fast, reliable, and nondestructive method for establishing the sameness of three different categories of injectable formulations, namely, Amphotericin B liposome for injection, enoxaparin injection, and iron sucrose injection. All three category formulations were evaluated in their native and artificially induced post degradation state to identify the discrimination power of the used instrumental techniques. The degradation of materials were confirmed by high performance liquid chromatography (HPLC). Based on the product category, pre and post-degradation samples were evaluated by selective instrumental methods like differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), fluorescence spectroscopy, particle size analysis by dynamic light scattering (DLS), small angle X-ray scattering (SAXS), Raman spectroscopy, inductively coupled plasma optical-emission spectrometry (ICP-OES) and circular dichroism study. All pre and post-degradation samples were further analyzed by TCS-PC. We observed that, TCS-PC can identify the differences between the initial and post degradation samples in very less time with promising discrimination power across the different injectable formulation types. Thus TCS-PC can be used as a fast and promising stability or sameness evaluation tool for different injectable drug products.

Detection of high-frequency gravitational waves using high-energy pulsed lasers

We propose a new method for detecting high-frequency gravitational waves (GWs) using high-energy pulsed lasers. Through the inverse Gertsenshtein effect, the interaction between a GW and the laser beam results in the creation of an electromagnetic signal. The latter can be detected using single-photon counting techniques. We compute the minimal strain of a detectable GW which only depends on the laser parameters. We find that a resonance occurs in this process when the frequency of the GW is twice the frequency of the laser. With this method, the frequency range 1013–1019 Hz is explored non-continuously for strains for current laser systems and can be extended to with future generation facilities.

Strong localization and suppression of Anderson modes in an asymmetrical optical waveguide.

In this paper, transverse Anderson localization of light waves in a 3D random network is achieved inside an asymmetrical type optical waveguide, formed within a fused-silica fiber by capillary process. Scattering waveguide medium originates from naturally formed air inclusions and Ag nanoparticles in rhodamine dye doped-phenol solution. Multimode photon localization is controlled by changing the degree of the disorder in the optical waveguide to suppress unwanted extra modes and obtain only one targeted strongly localized single optical mode confinement at the desired emission wavelength of the dye molecules. Additionally, the fluorescence dynamics of the dye molecules coupled into the Anderson localized modes in the disordered optical media are analyzed through time resolved experiments based on a single photon counting technique. The radiative decay rate of the dye molecules is observed to be enhanced up to a factor of about 10.1 through coupling into the specific Anderson localized cavity within the optical waveguide, providing a milestone for investigation of transverse Anderson localization of light waves in 3D disordered media to manipulate light-matter interaction.

Real time reconstruction of the fast electron spectrum from high intensity laser plasma interaction using gamma counting technique

X-ray and gamma fluxes from the high intensity laser-plasma interaction are extremely short, well beyond temporal resolution of any detectors. If laser pulses come repetitively, the single photon counting technique allows to accumulate the photon spectra, however, its relation to the spectrum of the initial fast electron population in plasma is not straightforward. We present efficient and fast approach based on the Geant4 package that significantly reduces computer time needed to re-construct the high energy tail of electron spectrum from experimental data accounting for the pileup effect. Here, we first tabulate gamma spectrum from monoenergetic electron bunches of different energy for a given experimental setup, and then compose the simulated spectrum. To account for the pileups, we derive analytical formula to reverse the data. We also consider errors coming from the approximation of the initial electron spectrum by the sum of monoenergetic impacts, the finite range of the electron spectrum, etc. and give estimates on how to choose modelling parameters to minimize the approximation errors. Finally, we present an example of the experimental data treatment for the case of laser-solid interaction using 50 fs laser pulse with intensity above 1018 W/cm2.

Quantifying the interactions of micellar drug delivery system components with albumin using time-resolved fluorescent methods.

The least requirement for the bioavailability of an active pharmaceutical ingredient (API) is its solubility in aqueous media. The solubilization of poorly soluble drugs is one of the key challenges of drug formulation development. Micellar drug delivery systems (MM-DDS) are efficient in solubilizing such APIs, are well tolerated by patients upon administration, are cost efficient and easy to produce. However, the number of marketed drugs is very limited. One problem identified by a reflection paper on this topic by the European Medicines Agency (EMA) is the number of different transformations of MM-DDS upon dilution by intravenous administration. The release of an API from MM-DDS and its subsequent binding to plasma proteins such as human serum albumin (HSA) determines the fate of the API in a human body. Tracking and characterization of such binding processes in a multicomponent system opens the door to understanding the drug - blood-components interaction. In this project we develop a label free time-resolved fluorescence tracking method to investigate the interaction of small molecules (API, MM-DDS components) with albumin. In detail, the data is collected using a time-correlated single-photon counting (TCSPC) technique at 280-nm excitation. Quenching of tryptophan fluorescence through Förster resonance energy transfer (FRET) to an API reveals information about the binding process. Affinity constants and stoichiometry can be obtained by global fitting of the titration curves and confirmed by the means of support plane analysis.

Unusual similarity of DNA solvation dynamics in high-salinity crowding with divalent cations of varying concentrations.

Double-stranded DNA bears the highest linear negative charge density (2e- per base-pair) among all biopolymers, leading to strong interactions with cations and dipolar water, resulting in the formation of a dense 'condensation layer' around DNA. Interactions involving proteins and ligands binding to DNA are primarily governed by strong electrostatic forces. Increased salt concentrations impede such electrostatic interactions - a situation that prevails in oceanic species due to their cytoplasm being enriched with salts. Nevertheless, how these interactions' dynamics are affected in crowded hypersaline environments remains largely unexplored. Here, we employ steady-state and time-resolved fluorescence Stokes shifts (TRFSS) of a DNA-bound ligand (DAPI) to investigate the static and dynamic solvation properties of DNA in the presence of two divalent cations, magnesium (Mg2+), and calcium (Ca2+) at varying high to very-high concentrations of 0.15 M, 1 M and 2 M. We compare the results to those obtained in physiological concentrations (0.15 M) of monovalent Na+ ions. Combining data from fluorescence femtosecond optical gating (FOG) and time-correlated single photon counting (TCSPC) techniques, dynamic fluorescence Stokes shifts in DNA are analysed over a broad range of time-scales, from 100 fs to 10 ns. We find that while divalent cation crowding strongly influences the DNA stability and ligand binding affinity to DNA, the dynamics of DNA solvation remain remarkably similar across a broad range of five decades in time, even in a high-salinity crowded environment with divalent cations, as compared to the physiological concentration of the Na+ ion. Steady-state and time-resolved data of the DNA-groove-bound ligand are seemingly unaffected by ion-crowding in hypersaline solution, possibly due to ions being mostly displaced by the DNA-bound ligand. Furthermore, the dynamic coupling of cations with nearby water may possibly contribute to a net-neutral effect on the overall collective solvation dynamics in DNA, owing to the strong anti-correlation of their electrostatic interaction energy fluctuations. Such dynamic scenarios may persist within the cellular environment of marine life and other biological cells that experience hypersaline conditions.

Structural isomerism induces pH dependent AIE-coupled ESIPT: an in-depth spectroscopic exploration with application in amine vapor sensing.

A novel excited state intramolecular proton transfer probe 3-(benzo[d]thiazol-2-yl)-4-hydroxy-5-methoxybenzaldehyde (3BTHMB) was synthesized and characterized using NMR, ESIMS and single crystal diffraction studies. The steady state and time resolved studies using the time correlated single photon counting (TCSPC) technique revealed that 3BTHMB shows ESIPT reaction with a time constant of 0.10-0.15 ns and longer local emission and anion emissions of time constants 0.5-2.0 ns and 3.0-4.0 ns, respectively, that co-exist in polar solution. In a polar protic solution like methanol, the ESIPT process is damped. Interestingly, 3BTHMB shows aggregation induced emission (AIE) solely in an acidic environment, evidenced by the enhanced quantum yield of emission (∼35%) in acidic solution as well as a long lifetime component of 7.4 ns that corresponds to the lifetime in the solid state. The AIE phenomenon was explored using the DLS technique where enhancement of the hydrodynamic radius in an acidic medium was observed. The judicious positioning of the formyl group in 3BTHMB gives the mentioned probe to show AIE in an acidic medium, as opposed to its previously reported structural isomer BTHMB, which showed no AIE phenomenon as well as its parent molecule 2-(benzo[d]thiazol-2-yl)-6-methoxyphenol (TMP), which showed AIE in neutral pH. The AIE property of 3BTHMB was exploited by successful and rapid detection of amine vapor in the solid state, qualitative ratiometric detection of aqueous pH, by observing the colour change under UV light in acidic (yellow-green) and basic to neutral (blue) medium. The above results pave the way for multiutility fluorescent probes for environmental purposes along with a deep understanding of the underlying photophysical processes which bestow the probes with such outstanding utilities.