Time-correlated Single Photon Counting Research Articles

Uncovering Integrated Dual-State ESIPT-Conductivity, Redox-Capacity, and Opto-Electronic Responses Toward Hg(II)/ Cr(III) of Aliphatic Fluorescent Polymers.

Excited-state intramolecular proton transfer (ESIPT)-associated dual-state emissive aliphatic dual-light emitting conducting polymers (DLECPs) having oxidation-reduction capacities are prepared polymerizing 2-acrylamido-2-methylpropane-1-sulfonic acid, methacrylic acid, and 2-methyl-3-(N-(2-methyl-1-sulfopropan-2-yl)acrylamido)propanoic acid monomers. Of as-synthesized DLECPs, nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies, fluorescent enhancements (I/I0), and computational investigation indicate intriguing photophysical features in DLECP3 (optimum composition). In DLECP3, ─CONH─, ─CON<, and ─COOH subluminophores are recognized by density-functional theory (DFT)/time-dependent-DFT calculations and experimental investigations. ESIPT-associated dual-state emission/conductivity, aggregation-enhanced emissions, selective opto-electronic responses toward Hg(II)/Cr(III) at 437/574nm, and redox properties of DLECP3 are supported by solid-state/solution spectroscopies, time-correlated single photon counting (TCSPC) measurements, dual-state excitation dependent emissions, microscopic images, electrochemical measurements, and DFT calculations. Here, preferential interaction of Hg(II)/Cr(III) with DLECP3 (amide)/DLECP3 (imidol) and reduction/oxidation of Hg(II)/Cr(III) to Hg(I)/Cr(VI) are substantiated by UV-vis, FTIR, and X-ray photoelectron spectroscopies; TCSPC measurements; NMR-titration; electrochemical studies; alongside computational calculations. The proton-electrical conductivities of DLECP3, Hg(II/I)-DLECP3, and Cr(III/VI)-DLECP3 in solids/solutions are 15.27 × 10-5/6.16 × 10-5, 19.60 × 10-5/25.52 × 10-5, and 26.69 × 10-5/27.60 × 10-5 S cm-1, respectively.

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.

Quantitative Analysis of Acquisition Speed of High-Precision FLIM Technologies via Simulation and Modeling

In practical applications such as cancer diagnosis and industrial detection, there is a critical demand for fast fluorescence lifetime imaging (Fast-FLIM). The Fast-FLIM systems suitable for complex environments are typically achieved by enhancing the hardware performance of time-correlated single-photon counting (TCSPC), with an acquisition speed of about a few frames per second (fps). However, due to the limitation of single-photon acquisition, the imaging speed is still far from the demand of practical application. The synchroscan streak camera (SC) maps signals from the temporal dimension to the spatial dimension, effectively overcoming the long acquisition time caused by single-photon acquisition. This paper constructs a method to calculate the acquisition time for the TCSPC-FLIM and SC-FLIM systems, and it quantitatively compares the speed. The research demonstrates that the main factors limiting the acquisition speed of the FLIM systems are the photon emission rate, the photon counting rate, the required SNR, the dwell time, and the number of parallel channels. In high-quality and large-scale lifetime imaging, the acquisition speed of the SC-FLIM is at least 104 times faster than that of the TCSPC-FLIM. Therefore, the synchroscan streak camera has more significant potential to promote Fast-FLIM.

Exploring Through-Space Charge Transfer-Mediated Optoelectrochemical Properties of Dual-State Luminescent Aliphatic Polymers and Optoelectronic Responses toward Metal Ions.

Herein, natural-synthetic hybrid dual-state luminescent conducting polymers (DLCPs/DLCP1-DLCP8) possessing significant optoelectrochemical properties are strategically developed by the polymerization of prop-2-enamide, cis-butenedioic acid, 2-acrylamido-2-methylpropane-1-sulfonic acid, and in situ-generated 2-(3-acrylamidopropanamido)-2-methylpropane-1-sulfonic acid alongside the grafting of gum tragacanth. The spectroscopic data of aliphatic DLCPs affirm DLCP7 as the most stable supramolecular assembly endowing optoelectronic properties. Computational calculations identified -C(═O)NH-, -C(═O)OH, -OH, and -SO3H as subluminophores. The absorption spectra, excitation wavelength-/solvent-polarity-/concentration-dependent luminescence, solid state luminescence, aggregation-induced enhanced luminescence, and time-correlated single photon count (TCSPC) studies confirm the occurrence of aggregation-mediated intramolecular through-space charge transfer (ITSCT) in the excited state of DLCP7. Mulliken charge, natural bond orbital, dipole moments, and electronic potential surface analyses confirm the charge donor-acceptor system in DLCP7. Furthermore, the selective optoelectronic response of DLCP7 toward Ca2+/Cu(II) at 438/574 nm is explored using ultraviolet-visible spectra, TCSPC analyses, a dynamic light scattering study, and computational investigations. The chelation-enhanced luminescence and ITSCT inhibition are responsible for turn-on and turn-off detections of Ca2+ and Cu(II), respectively. Cu(II) → Cu(I) reduction in a DLCP7 solution is inferred from electrochemical and spectroscopic analyses. The conductivities of 9.65 × 10-5 S cm-1 (solid state) and 44.35 × 10-5 S cm-1 (solution) in DLCP7 are validated by current-voltage and electrochemical impedance measurements. Again, strong electronic conductivities of 43.89 × 10-5 S cm-1 (solid state)/53.34 × 10-5 S cm-1 (solution) and 45.42 × 10-5 S cm-1 (solid state)/64.81 × 10-5 S cm-1 (solution) are observed in Ca2+-DLCP7 and Cu(II)-DLCP7, respectively.

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.

Synthesis and optimization of chitosan-incorporated semisynthetic polymer/α-Fe2O3 nanoparticle hybrid polymer to explore optimal efficacy of fluorescence resonance energy transfer/charge transfer for Co(II) and Ni(II) sensing

Initially, four synthetic fluorescent polymers (SFPs) are synthesized from α-methacrylic acid and methanolacrylamide monomers carrying –C(=O)OH and –C(=O)NH subfluorophores, respectively. Among SFPs, ∼1:1 incorporation of subfluorophores in the optimum SFP3 is explored by spectroscopic analyses. Subsequently, chitosan is incorporated in SFP3 to produce five semi-synthetic fluorescent polymers (SSFPs). The maximum incorporation of chitosan in SSFP4 is supported by different spectroscopies. In SSFP4, strong electrostatic interactions among polar functionalities of chitosan and synthetic polymer favor resonance-associated charge transfer (RCT) from SSFP4-(amide) to SSFP4-(canonical). Finally, three hybrid fluorescent polymers (HFPs) are fabricated encapsulating iron-oxide nanoparticle within SSFP4. The maximum proportion of hematite (α-Fe2O3) phase in HFPs is explored by spectroscopic, magnetometric, microscopic, and light scattering studies. HFP2 shows local/RCT/fluorescence resonance energy transfer (FRET) emission at 393/460/570 nm. In HFP2, FRET, RCT, and ratiometric pH-sensing within 3.0–6.5 phenomena are explored by solvent polarity effects, time-correlated single photon counting, quantum yield measurements, alongside I431/I460 vs pH plots. RCT and FRET emissions of HFP2 are utilized for selective sensing of Co(II)/Ni(II) with limits of detection of 4.990 ppb (460 nm)/4.353 ppb (570 nm) and 45.041 ppb (428 nm)/29.617 ppb (527 nm) in organic and aqueous solutions, respectively.

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC).

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is, however, a growing gap between the state of the art and what is accessible to biologists, biochemists, medical researchers, or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed microspectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy, time-correlated single-photon counting and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines, are provided, to allow non-optics specialists to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.

On the glow of cremated remains: long-lived green photo-luminescence of heat-treated human bones.

The long-lived green luminescence of human bone (that has been heated to 600°C for a short duration) is attributed to a carbon quantum dot material (derived from collagen) encapsulated and protected by an inorganic matrix (derived from bone apatite) and is more intense in dense rigid and crystalline parts of (healthy) human bones. The strong collagen-apatite interaction results (upon decomposition) in a protective inorganic environment of the luminescent centers allowing long-lived triplet-based emission of a carbon (quantum) dot-like material at room temperature, as well as resilience against oxidation between 550 and 650°C. The graphitic black phase (obtained upon heating around 400°C) is a precursor to the luminescent carbon-based material, that is strongly interacting with the crystalline inorganic matrix. Human bone samples that have been heated to 600°C were subjected to steady-state and time-resolved spectroscopy. Excitation-emission matrix (EEM) luminescence spectroscopy revealed a broad range of excitation and emission wavelengths, indicating a heterogeneous system with a broad density of emissive states. The effect of low temperature on the heat-treated bone was studied with Cryogenic Steady State Luminescence Spectroscopy. Cooling the bone to 80K leads to a slight increase in total emission intensity as well as an intensity increase towards to red part of the spectrum, incompatible with a defect state model displaying luminescent charge recombination in the inorganic matrix. Time-resolved spectroscopy with an Optical Multichannel Analyzer (OMA) and Time Correlated Single Photon Counting (TCSPC) of these samples showed that the decay could be fitted with a multi-exponential decay model as well as with second-order decay kinetics. Confocal Microscopy revealed distinct (plywood type) structures in the bone and high intensity-fast decay areas as well as a spatially heterogeneous distribution of green and (fewer) red emissive species. The use of the ATTO 565 dye aided in bone-structure visualization by chemical adsorption. Conceptually our data interpretation corresponds to previous reports from the material science field on luminescent powders.

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.

Room-Temperature Solution Fluorescence Excitation Correlation Spectroscopy.

Single-molecule fluorescence spectroscopy is a powerful tool for investigating the physical properties of individual molecules, yet elucidating the fast fluctuation dynamics of freely diffusing single molecules in solution at room temperature, where a variety of chemical and biological processes occur, remains challenging. In this study, we report on fluorescence excitation correlation spectroscopy of room-temperature solutions, which enables the study of spontaneous fluctuation of the excitation spectrum with microsecond time resolution. By employing Fourier transform spectroscopy with broadband femtosecond pulses and time-correlated single-photon counting, we achieved fluorescence excitation spectroscopy of a room-temperature solution at the single-molecule level. Building upon this single-molecule measurement, we obtained an excitation wavelength-resolved fluorescence autocorrelation function in the microsecond to millisecond range, demonstrating the potential of this method to elucidate fast, spontaneous, time-dependent changes of excitation spectra in statistically equilibrated systems. With further development, this method will allow the study of spectral exchange associated with transitions between sub-ensembles of solution-phase molecules with unprecedented time resolution.

Integrated Active Quenching Circuit for high-rate and distortionless SPAD-based time-resolved fluorescence applications.

Time-Correlated Single Photon Counting (TCSPC) is a pivotal technique in low-light-detection applications, renowned for its exceptional sensitivity and bandwidth, widely used in Fluorescence Lifetime Imaging Microscopy (FLIM) and quantum optics. Despite its features, TCSPC is significantly hindered by the pile-up effect, which may distort measurements at high photon-detection rates. Overcoming pile-up is challenging, with traditional solutions often involving complex post-processing or multichannel systems, complicating the TCSPC setup and limiting performance. A breakthrough to overcome this issue is matching the photodetector dead time to an integer multiple of the laser period, obtaining a distortionless histogram even at high illumination conditions. Building on this concept, we present an Active Quenching Circuit (AQC) developed in high-voltage 150 nm technology, achieving unprecedented control over the Single Photon Avalanche Diode (SPAD) dead time. Our design compensates for Process, Voltage, and Temperature (PVT) variations, ensuring ultra precise and robust dead time tuning. The presented AQC achieves a dead-time resolution of 50 ps suitable for time-resolved experiments within a selectable range of laser frequencies from 20 to 100 MHz, maintaining close-to- ideal linearity in dead-time control. Experimental validations through fluorescence measurements reveal a distortion as low as 0.43% under elevated count-rate conditions, highlighting the efficacy of our circuit in overcoming the pile-up limitation.

Dependence of photophysical properties on stereoisomers: The case of nonsymmetric 2-aryl(benz)imidazole-annulated derivatives

Nonsymmetric 2-aryl (benz)imidazole derivatives were previously synthesized via C-H/N-H annulation of imidazole derivatives from lapachol moieties with internal alkynes as an efficient tool to increase the π-system of lapimidazole derivatives. To investigate their photophysical properties, time-resolved and steady-state fluorescence measurements were carried out on dropcast film, powder, and diluted solution. Despite having the same number of single and double bonds, slight differences in their structures lead to distinct photophysical properties. The time-correlated single-photon counting measurements also demonstrated that the aspects of the temporal decay curves are dependent on the treatment of the samples during the measurements. The decay curves were relatively well fitted using an alternative method based on the exponentially modified Gaussian function, giving rise to a more complete set of fitting parameters. Finally, theoretical calculations corroborate that small structural changes in molecules can significantly alter their conformational dynamics, electronic spectra, and overall electronic behavior and stability.

Improved echo signal detection for pseudo-random single-photon counting laser ranging

In this paper, a new echo signal detection method, to the best of our knowledge, for pseudo-random single-photon counting ranging (PSPCR) LiDAR systems is proposed, which is applied for long distances, low repetition rates, and system cost reduction. First, in order to achieve a comparable temporal resolution as that in time-correlated single-photon counting (TCSPC) systems, we extend the pseudo-random code to discriminate the minimal time slot in time correlation. Second, we use the full width at half maxima (FWHM) in the duration of each pseudo-random code for correlation to reduce the impact of pulse width variation and timing jitters on ranging accuracy. Third, we study the bias and errors caused by using synchronous signals as the “START” signal, and propose to use the time of flight (ToF) at half energy to reduce the walk error. Simulation results show that, compared with existing PSPCR methods, the proposed method improves ranging accuracy with a lower repetition rate and lower peak and average power—centimeter-level ranging accuracy over tens of kilometers can be achieved using a laser with a repetition rate of 400 kHz, peak power of up to 1 kW, and average power of up to 1 W.

Timing limits of ultrafast cross-luminescence emission in CsZnCl-based crystals for TOF-CT and TOF-PET

BackgroundGood timing resolution in medical imaging applications such as TOF-CT or TOF-PET can boost image quality or patient comfort significantly by reducing the influence of background noise. However, the timing resolution of state-of-the-art detectors in CT and PET are limited by their light emission process. Core-valence cross-luminescence is an alternative, but well-known compounds (e.g. BaF2) pose several problems for medical imaging applications, such as their emission wavelength in the deep UV. CsZnCl-based materials show promise to solve this issue, as they provide fast decay times of 1–2 ns and an emission wavelength around 300 nm.ResultsIn this work, we investigated two CsZnCl-compounds: Cs2ZnCl4 and Cs3ZnCl5. We validated the previously published decay times on a time-correlated single-photon counting setup with 1.786 ± 0.016 ns for Cs2ZnCl4 and 1.034 ± 0.013 ns for Cs3ZnCl5. The setup’s high resolution enabled the discovery of an additional prompt emission component with a significant abundance of 98 ± 18 (Cs2ZnCl4) and 86 ± 14 (Cs3ZnCl5) photons/MeV energy deposit. In a PET coincidence experiment, we measured the best coincidence time resolution (CTR) of 62 ps (FWHM) for Cs2ZnCL4 coupled to FBK VUV SiPMs with silicon oil. To assess the CTR for lower energies, we filtered the energy along the Compton continuum and found a deteriorated CTR that seems to be mainly influenced by photon statistics. Furthermore, this study gave us a rough estimate of e.g. 150 ps (FWHM) CTR at 100 keV energy for Cs2ZnCL4. From measurements with high activity of 14 MBq to check for pile-up effects we assume that Cs2ZnCl4 is better suited for high-rate time-of-flight applications than lutetium-based oxides. Simulations demonstrated that the stopping power of Cs2ZnCl4 is lower than for LSO:Ce,Ca, meaning that a high amount of material would be needed for TOF-PET applications. However, the stopping power seems acceptable for applications in TOF-CT.ConclusionsThe fast decay time, state-of-the-art CTR in benchtop experiments and high-rate suitability make CsZnCl materials a promising candidate for time-of-flight experiments. We consider especially TOF-CT a suitable application due to its relatively low X-ray energies (~ 100 keV) and the thusly acceptable stopping power of Cs2ZnCl4. Currently, further exploration of the prompt emission and its creation mechanism is planned, as well as investigating the light transport of Cs2ZnCl4 in longer crystals.

Fluorescence Lifetime Measurement For Studying Evaporation Of Bi-Component Droplet

Sprays are essential in engineering applications like evaporators and combustion chambers. Predicting the evaporation of multi-component liquid droplets is complex due to changing composition and properties. Measuring droplet composition is challenging, with limited non-intrusive methods available. Raman spectroscopy faces hurdles like weak signals and interference. Intensity based Laser-Induced Fluorescence (LIF) requires optical models to interpret data due to varying droplet size and position. Fluorescent dyes in liquid mixtures are sensitive to temperature, complicating measurements. Fluorescence lifetime measurements offer a promising alternative, being an absolute quantity less affected by reabsorption. This study develops a novel method using fluorescence lifetime to analyze droplet composition, leveraging Time-Correlated Single Photon Counting (TCSPC) for precise measurements. Factors such as dye concentration, solvent polarity, and temperature sensitivity are investigated. Calibration curves for ethanol-water mixtures correlate fluorescence lifetime with volume fraction. Experimental setups use TCSPC and femtosecond lasers for dye excitation. Results show that fluorescence lifetime decreases linearly with solvent polarity and is influenced by temperature and dye concentration. High dye concentrations cause self-quenching, altering temperature sensitivity. The method was applied to study the evaporation of ethanol-water droplets under acoustic levitation. Findings indicate that evaporation rates slow as the water fraction increases, with initial measurements showing higher water content due to partial evaporation. This research introduces a novel technique for droplet compositional analysis using fluorescence lifetime, providing insights into optimizing measurement accuracy and advancing understanding in engineering and related fields.

Exploring the molecular binding mechanism of 6-fluoro, 4-hydroxy, 2- methyl quinoline with TiO2 nanoparticles: A spectroscopic, thermodynamic, and insights into the solvatochromic effect.

This study investigates the interaction between titanium oxide nanoparticles (TiO2 NPs) and the heterocyclic fluorophore 6-fluoro,4-hydroxy,2-methylquinoline (6-FHMQ), aiming to understand fluorescence quenching mechanisms and thermodynamic characteristics. Spectroscopic techniques including spectrofluorometry (FL) and spectrophotometry (UV-Vis) were used, with a lifetime decay (τ) of 0.18ns for 6-FHMQ measured using time correlated single photon counting (TCSPC). The interaction between 6-FHMQ and TiO2 NPs revealed a mix of static and dynamic fluorescence quenching mechanisms, with increasing quenching constants (Ksv) and a higher bimolecular quenching rate constant (Kq). The dynamic nature was highlighted by a temperature-dependent increase in binding sites from 1 to ~ 2. Spontaneous complexation was affirmed by negative change in free energy (ΔG), with negative change in enthalpy (ΔH) and a positive change in entropy (ΔS) values indicating favorable electrostatic and ionic interactions. The impact of varying TiO2 NP concentrations on 6-FHMQ absorption was analyzed using the Benesi-Hildbrand equation, with a quantum yield of 0.61 determined. By forster resonance energy transfer (FRET) theory, the proximity between 6-FHMQ and TiO2 NPs was found to be less than 70Å. Ground and excited state dipole moments of 6-FHMQ in different solvents were calculated to demonstrate solvent sensing ability and charge transfer properties. Ultimately, this study serves as a testament to the power of scientific innovation in the realms of drug delivery and tissue engineering.

Synthesis of Intrinsically-Fluorescent Aliphatic Tautomeric Polymers for Proton-Conductivity, Dual-State Emission, and Sensing/Oxidation-Reduction of Metal Ions.

Herein, fluorescent conducting tautomeric polymers (FCTPs) are developed by polymerizing 2-methylprop-2-enoic acid (MPEA), methyl-2-methylpropenoate (MMP), N-(propan-2-yl)prop-2-enamide (PPE), and in situ-anchored 3-(N-(propan-2-yl)prop-2-enamido)-2-methylpropanoic acid (PPEMPA). Among as-synthesized FCTPs, the most promising characteristics in FCTP3 are confirmed by NMR and Fourier transform infrared (FTIR) spectroscopies, luminescence enhancements, and computational studies. In FCTP3, ─C(═O)NH─, -C(═O)N<, ─C(═O)OH, and ─C(═O)OCH3 subluminophores are identified by theoretical calculations and experimental analyses. These subluminophores facilitate redox characteristics, solid state emissions, aggregation-enhanced emissions (AEEs), excited-state intramolecular proton transfer (ESIPT), and conductivities in FCTP3. The ESIPT-associated dual emission/AEEs of FCTP3 are elucidated by time correlated single photon counting (TCSPC) investigation, solvent polarity effects, concentration-dependent emissions, dynamic light scattering (DLS) measurements, field emission scanning electron microscopy images, and computational calculations. The cyclic voltammetry measurements of FCTP3 indicate cumulative redox efficacy of ─C(═O)OH, ─C(═O)NH─/-C(═O)N<, ─C(─O─)═NH+─/─C(─O─)═N+, and ─C(═N)OH functionalities. In FCTP3, ESIPT-associated dual-emission enable in the selective detection of Cr(III)/Cu(II) at λem1/λem2 with the limit of detection of 0.0343/0.079 ppb. The preferential interaction of Cr(III)/Cu(II) with FCTP3 (amide)/FCTP3 (imidol) and oxidation/reduction of Cr(III)/Cu(II) to Cr(VI)/Cu(I) are further supported by NMR-titration; FTIR and X-ray photoelectron spectroscopy analyses; TCSPC/electrochemical/DLS measurement; alongside theoretical calculations. The proton conductivity of FCTP3 is explored by electrochemical impedance spectroscopy and I-V measurements.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Acridinedione-phthalimide conjugates: Intramolecular electron transfer and singlet oxygen generation studies for optical and photodynamic therapy applications

We reported herein the synthesis, characterization of hybrid conjugates composed of phthalimide (Phth) and acridine-1,8-diones (Acr) for optical and medical applications. For the synthetic procedure, a three-step synthetic strategy has been utilized. The optical properties of the examined 1,8-acridinedione–phthalimide connected molecules (AcrPhth 1–5) have been examined utilizing various spectroscopic techniques, e.g., steady-state absorption and fluorescence, and time-correlated single photon counting. The steady-state absorption studies showed that AcrPhth 1–5 absorbs the light in the UV and visible region. The fluorescence studies of AcrPhth 1–5 exhibited significant fluorescence quenching compared to the acridinedione control compounds (Acr 1–5) suggesting the occurrence of electron-transfer reactions from the electron donating acridinedione moiety (Acr) to the electron accepting phthalimide moiety (Phth). The rate and efficiency of the electron-transfer reactions were determined from the fluorescence lifetime measurements indicating the fast electron-transfer processes of the covalently connected AcrPhth 1–5 conjugates. Computational studies supported the intramolecular electron-transfer reaction of AcrPhth conjugates using ab initio B3LYP/6-311G methods. In the optimized structures, the HOMO was found to be entirely located on the Acr entity, while the LUMO was found to be entirely on the Phth entity. Further, the synthesized compounds were tested as photosensitizers for generating the singlet oxygen species, which is a key factor in the photodynamic therapy (PDT) applications. The nanosecond laser flash measurements enable us to detect the triplet-excited states of examined Acr and AcrPhth conjugates, determining the triplet quantum yields, and direct detecting the singlet oxygen in an accurate way. From this observation, the singlet quantum yields were found to be in the range of 0.12–0.27 (for Acr 1–5) and 0.07–0.19 (for AcrPhth 1–5 conjugates). The molecular docking studies revealed that compound AcrPhth 2 exhibited high binding affinity with for key genes (p53, TOP2B, p38, and EGFR) suggesting its potential as a targeted anticancer therapy.Graphical abstractEfficient intramolecular electron-transfer reaction for the light harvesting conjugates! In addition, the conjugates can be used as good photosensitizers for PDT