Fluorescence Decay Kinetics Research Articles

Anti-Stokes fluorescence excitation reveals conformational mobility of the C-phycocyanin chromophores.

The structural organization of natural pigment-protein complexes provides a specific environment for the chromophore groups. Yet, proteins are inherently dynamic and conformationally mobile. In this work, we demonstrate the heterogeneity of chromophores of C-phycocyanin (C-PC) from Arthrospira platensis. Part of the population of trimeric C-PC is subject to spontaneous disturbances of protein–protein interactions resulting in increased conformational mobility of the chromophores. Upon fluorescence excitation in the visible range, the spectral signatures of these poorly populated states are masked by bulk chromophore states, but the former could be clearly discriminated when the fluorescence is excited by near-infrared quanta. Such selective excitation of conformationally mobile C-PC chromophores is due to the structure of their S1 level, which is characterized by a significantly broadened spectral line. We demonstrate that the anti-Stokes C-PC fluorescence is the result of single-photon absorption. By combining spectral and structural methods, we characterize four distinct states of C-PC chromophores emitting at 620, 650, 665, and 720 nm and assigned the fast component in the anti-Stokes fluorescence decay kinetics in the range of 690–750 nm to the chromophores with increased conformational mobility. Our data suggest that the spectral and temporal characteristics of the anti-Stokes fluorescence can be used to study protein dynamics and develop methods to visualize local environment parameters such as temperature.

Substituted boron subphthalocyanines – Prospective compounds for theranostics: Synthesis, photochemical properties and in vitro cytotoxicity

Boron subphthalocyanines bearing a wide range of functional groups were tested as prospective photosensitizers and fluorophores. Synthetic protocols to novel 4-ethoxycarbonylphenoxy-substituted subphthalocyanine and phenoxy-substituted subphthalocyanine boron phenolate were presented. Differential triplet-triplet absorption spectra of subphthalocyanines were registered and triplet state lifetimes (τT = 0.6 ÷ 3.1 ms) were obtained. The results of flash photolysis measurements predicted the good ability of target subphthalocyanines to produce singlet oxygen and it was supported by the measurements of 1O2 quantum yields (ΦΔ = 0.47 ÷ 0.85). At the same time, satisfactory yields of fluorescence (Φf = 0.13 ÷ 0.50) were observed for most of the subphthalocyanines. The fluorescence decay kinetics were studied to determine the fluorescence lifetimes (τf = 0.30 ÷ 3.05 ns) and confirm the regularities obtained for fluorescence yields. Pronounced fluorescent properties allowed investigating the distribution of subphthalocyanines in the endoplasmic reticulum. In addition, the binding constants with human serum albumin were determined. tert-Butyl- and iodo-substituted subphthalocyanines demonstrated remarkable photocytotoxicity in human epidermoid carcinoma cells at micromolar concentrations. At the same time, target boron subphthalocyanines exhibited low dark cytotoxicity. All mentioned above make boron subphthalocyanines promising candidates as highly effective photosensitizers for photodynamic therapy and fluorescent labels for diagnostic.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase.

Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F- ) and chloride (Cl- ). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl- and F- bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl- binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F- binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity.

Circular Permutants of BrUSLEE Protein as Fluorescent pH Indicators

—Two variants of circularly permuted BrUSLEE, a green fluorescent protein with a short fluorescence lifetime, have been engineered. We characterized the pH-dependence of fluorescence decay kinetics of these fluorophores. It was shown that both permutants (cpBrUS and cpBrUS-145) exhibit three-component fluorescence decay kinetics, with the lifetime of the one component varying within the ~3000–300 ps range upon pH shift from 5.5 to 9.0. At the same time, the original BrUSLEE does not show a significant change in the fluorescence decay kinetics within the physiologically relevant pH-range of 6.0–8.5. The described pH-dependence allows considering the BrUSLEE permutants as pH indicators with the fluorescence lifetime readout.

Photobiology of lipofuscin granules in the retinal pigment epithelium cells of the eye: norm, pathology, age.

Lipofuscin granules (LGs) are accumulated in the retinal pigment epithelium (RPE) cells. The progressive LG accumulation can somehow lead to pathology and accelerate the aging process. The review examines composition, spectral properties and photoactivity of LGs isolated from the human cadaver eyes. By use of atomic force microscopy and near-field microscopy, we have revealed the fluorescent heterogeneity of LGs. We have discovered the generation of reactive oxygen species by LGs, and found that LGs and melanolipofuscin granules are capable of photoinduced oxidation of lipids. It was shown that A2E, as the main fluorophore (bisretinoid) of LGs, is much less active as an oxidation photosensitizer than other fluorophores (bisretinoids) of LGs. Photooxidized products of bisretinoids pose a much greater danger to the cell than non-oxidized one. Our studies of the fluorescent properties of LGs and their fluorophores (bisretinoids) showed for the first time that their spectral characteristics change (shift to the short-wavelength region) in pathology and after exposure to ionizing radiation. By recording the fluorescence spectra and fluorescence decay kinetics of oxidized products of LG fluorophores, it is possible to improve the methods of early diagnosis of degenerative diseases. Lipofuscin ("aging pigment") is not an inert "slag". The photoactivity of LGs can pose a significant danger to the RPE cells. Fluorescence characteristics of LGs are a tool to detect early stages of degeneration in the retina and RPE.

Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.

The first encoded SARS-CoV-2 protein (Nsp1) binds to the human 40S ribosome and blocks synthesis of host proteins, thereby inhibiting critical elements of the innate immune response. The final 33 residues of the natively unstructured Nsp1 C-terminus adopt a helix-turn-helix geometry upon binding to the ribosome. We have characterized the fluctuating conformations of this peptide using circular dichroism spectroscopy along with measurements of tryptophan fluorescence and energy transfer. Tryptophan fluorescence decay kinetics reveal that copper(II) binds to the peptide at micromolar concentrations, and electron paramagnetic resonance spectroscopy indicates that the metal ion coordinates to the lone histidine residue.

Selective laser spectroscopy of a highly dispersed Bi4Ge3O12 scintillator doped with Pr3+ ions

The optical properties of a highly dispersed bismuth orthogermanate powder Bi4Ge3O12: Pr3+ (HDP_BGO: Pr3+) were studied at Т = 300 K. The fluorescence of undoped HDP_BGO under ultraviolet (UV) excitation (λexc = 266 nm) was shown to result from Frenkel excitons (GeO44−, BiO69−). The HDP_BGO fluorescence decay kinetics under UV excitation had a complex decay form similar to that of BGO single crystal and was a sum of four exponentials with lifetimes τ1, 2, 3, 4– 0.16/0.30/0.70/2.7 μs respectively. Two types of Pr3+ ions optical centers with lifetimes of ∼5.0 and 85 μs have been determined in a HDP_BGO: Pr3+. The resonance energy transfer of the electronic excitation from the HDP_BGO to Pr3+ions was identified. The effect of Pr3+ ions on the HDP_BGO fluorescence lifetime reduction from 160 to 35 ns was established.

Optical and visible and near-infrared fluorescence aspects of Er3+, Tm3+, and Nd3+-doped B2O3-rich glasses for fiber amplifiers and NIR lasers

For synthesized each 1 mol% Er3+, Tm3+, and Nd3+-doped borate-rich glasses, optical and luminescence traits including fluorescence decay dynamics were explored. For Er3+: glass, at 1.4–1.65 μm range a wide NIR (near-infrared) luminescence band centered at 1.531 μm (4I13/2 → 4I15/2) and in Tm3+: glass, at 1.35–1.6 μm span a broad NIR fluorescence band centered at 1.458 μm (3H4→3F4) are obtained by 980 nm LD (laser diode) pumping (4I15/2 → 4I11/2) and 808 nm LD excitation (3H6→3H4) separately. Under 808 nm LD pumping (4I9/2 → 4F5/2+2H9/2), in Nd3+: glass, three emission bands peaked at 0.901 μm (4F3/2 → 4I9/2), 1.057 μm (4F3/2 → 4I11/2), and 1.329 μm (4F3/2 → 4I13/2) respectively are observed and in all such emissions, comparably the one obtained from transition 4F3/2 → 4I11/2 is highly intense. For acquired main visible and NIR emissions, Δλeff (effective bandwidth), by Füchtbauer–Ladenburg approach σemi (stimulated emission cross-section), gain bandwidth, and optical gain are evaluated. 75.73 nm, 113.67 nm, and 57.37 nm are the Δλeff values for Er3+: 4I13/2 → 4I15/2, Tm3+: 3H4→3F4, and Nd3+: 4F3/2 → 4I13/2 transitions accordingly. Using McCumber's theory, the calculated peak σemi for Er3+: 4I13/2 → 4I15/2 and Nd3+: 4F3/2 → 4I9/2 transitions are 1.495 × 10–20 cm2 and 0.5556 × 10–20 cm2, respectively. 4I13/2 → 4I15/2 transition gain cross-section spectra reveals C-band amplification at P (population inversion) = 50% in Er3+: glass, whereas at P = 30% for 4F3/2 → 4I9/2 transition gain cross-section is positive for lasing action at ∼0.9 μm in Nd3+: glass. All analyzed NIR luminescence outcomes reveal that studied glasses could be potential gain media for C-, S-, and O-bands fiber amplifiers and solid-state lasers.

Fabrication and spectroscopic properties of hot-pressed Tb:CaF2 transparent ceramics with different doping concentrations

High quality of x at.% Tb:CaF2 (x = 1, 3, 5, 7, 9) transparent ceramics prepared by hot-pressed sintering achieved an average transmittance at 1500 nm of 88%. The effect of Tb3+ concentrations on the phase composition, microstructure, emission spectra, and fluorescence decay dynamics of Tb:CaF2 transparent ceramics were investigated. XRD and FESEM results demonstrated that the incorporation of Tb3+ did not cause significant changes in the crystal structure of CaF2, and the grain size gradually decreased with increasing Tb3+ doping concentration. Under the excitation of 350 nm, the strongest emission peak was located at 542 nm, which was attributed to the 5D4→7F5 transition of Tb3+. The green emission intensity enhanced linearly with increasing Tb3+ concentration from 1 at.% to 9 at.%. The fluorescence lifetime of 5D4 level was also investigated, and the values were comparable to the corresponding single crystals. All the results suggest that Tb:CaF2 transparent ceramic is a kind of potential green luminescence material.

Automated Phasor Segmentation of Fluorescence Lifetime Imaging Data for Discriminating Pigments and Binders Used in Artworks.

The non-invasive analysis of fluorescence from binders and pigments employed in mixtures in artworks is a major challenge in cultural heritage science due to the broad overlapping emission of different fluorescent species causing difficulties in the data interpretation. To improve the specificity of fluorescence measurements, we went beyond steady-state fluorescence measurements by resolving the fluorescence decay dynamics of the emitting species through time-resolved fluorescence imaging (TRFI). In particular, we acquired the fluorescence decay features of different pigments and binders using a portable and compact fibre-based imaging setup. Fluorescence time-resolved data were analysed using the phasor method followed by a Gaussian mixture model (GMM) to automatically identify the populations of fluorescent species within the fluorescence decay maps. Our results demonstrate that this approach allows distinguishing different binders when mixed with the same pigment as well as discriminating different pigments dispersed in a common binder. The results obtained could establish a framework for the analysis of a broader range of pigments and binders to be then extended to several other materials used in art production. The obtained results, together with the compactness and portability of the instrument, pave the way for future in situ applications of the technology on paintings.

Effect of nanodiamonds on fluorescence enhancement of tryptophan phototransformation reaction products in the presence of halocarbons

The role of nanodiamonds in the reaction of photochemical transformation of tryptophan in the presence of halogen hydrocarbons has been studied. The photochemical transformation of free tryptophan in a suspension with diamond nanoparticles and in a hybrid complex with them when exposed to UV radiation in the presence of chloroform is investigated. Data from stationary and time-resolved spectroscopic studies show the presence of non-radiative transfer of electron excitation energy between tryptophan molecules and its photodestruction products for the case of a covalent complex with a nanodiamond. It is shown that in the presence of energy transfer, an increase in the intensity of integral fluorescence occurs in the range of ~ 450 nm. Thus, a covalent tryptophan complex with a nanodiamond can serve as a fluorescent marker for the presence of chloroform in solution. Keywords: nanoscale diamonds, hybrid complexes, tryptophan phototransformation, spectral-luminescent properties, fluorescence decay kinetics, chloroform.

Nonadiabatic excited-state dynamics of ReCl(CO)3(bpy) in two different solvents.

We present a study of excited-states relaxation of the complex ReCl(CO)3(bpy) (bpy = 2,2-bipyridine) using a nonadiabatic TD-DFT dynamics on spin-mixed potential energy surfaces in explicit acetonitrile (ACN) and dimethylsulfoxide (DMSO) solutions up to 800 fs. ReCl(CO)3(bpy) belongs to a group of important photosensitizers which show ultrafast biexponential subpicosecond fluorescence decay kinetics. The choice of solvents was motivated by the different excited-state relaxation dynamics observed in subpicosecond time-resolved IR (TRIR) experiments. Simulations of intersystem crossing (ISC) showed the development of spin-mixed states in both solvents. Transformation of time-dependent populations of spin-mixed states enabled to monitor the temporal evolution of individual singlet and triplet states, fitting of bi-exponential decay kinetics, and simulating the time-resolved fluorescence spectra that show only minor differences between the two solvents. Analysis of structural relaxation and solvent reorganization employing time-resolved proximal distribution functions pointed to the factors influencing the fluorescence decay time constants. Nonadiabatic dynamics simulations of time-evolution of electronic, molecular, and solvent structures emerge as a powerful technique to interpret time-resolved spectroscopic data and ultrafast photochemical reactivity.

Temperature-sensitive fluorescence decay kinetics of thioflavin T derivatives in glycerol

Possible use of Thioflavin T based fluorescent molecular rotors as temperature sensors was assessed in this work. Fluorescent properties of Thioflavin T and its derivative 6-Me-BTA-2C in 99% glycerol were studied using steady-state and time-resolved fluorescence spectroscopy methods. For Thioflavin T in glycerol it has been found that temperature growth from 261 K to 353 K results in ~ 3 orders of magnitude increase of non-radiative decay rate constant knr for the excited state of the molecule. Fluorescence decay studies by time-correlated single photon counting method showed that fluorescence decay kinetics of Thioflavin T in glycerol can be used for temperature measurements in the range 260-290 K. For 6-Me-BTA-2C molecule the range of the maximal sensitivity was shifted to higher temperatures (280-320 K). The obtained results about fluorescent properties and decay kinetics of Thioflavin T based dyes in highly viscous media can be used to develop nanoscale temperature sensors. Keywords: temperature sensor, fluorescent molecular rotor, nanothermometry, thioflavin T, TICT.

Температурная зависимость кинетики затухания флуоресценции производных тиофлавина T в глицерине

Possible use of Thioflavin T based fluorescent molecular rotors as temperature sensors was assessed in this work. Fluorescent properties of Thioflavin T and its derivative 6-Me-BTA-2C in 99 % glycerol were studied using steady-state and time-resolved fluorescence spectroscopy methods. For Thioflavin T in glycerol it has been found that temperature growth from 261 K to 353 K results in ~3 orders of magnitude increase of non-radiative decay rate constant k_nr for the excited state of the molecule. Fluorescence decay studies by time-correlated single photon counting method showed that fluorescence decay kinetics of Thioflavin T in glycerol can be used for temperature measurements in the range 260–290 K. For 6-Me-BTA-2C molecule the range of the maximal sensitivity was shifted to higher temperatures (280–320 K). The obtained results about fluorescent properties and decay kinetics of Thioflavin T based dyes in highly viscous media can be used to develop nanoscale temperature sensors.

Analysis of Fluorescence Decay Kinetics of Indocyanine Green Monomers and Aggregates in Brain Tumor Model In Vivo.

Spectroscopic approach with fluorescence time resolution allows one to determine the state of a brain tumor and its microenvironment via changes in the fluorescent dye’s fluorescence lifetime. Indocyanine green (ICG) is an acknowledged infra-red fluorescent dye that self-assembles into stable aggregate forms (ICG NPs). ICG NPs aggregates have a tendency to accumulate in the tumor with a maximum accumulation at 24 h after systemic administration, enabling extended intraoperative diagnostic. Fluorescence lifetime analysis of ICG and ICG NPs demonstrates different values for ICG monomers and H-aggregates, indicating promising suitability for fluorescent diagnostics of brain tumors due to their affinity to tumor cells and stability in biological tissue.

Altering the distribution of excited-state lifetimes in aminated GFP chromophores by Ag nanohole arrays

Fluorescence of the modified GFP chromophore diethyl-ABDI-BF2 dispersed in PMMA matrix is studied on top of glass, continuous and perforated optically thin silver films. In polymer, the fluorescence decay kinetics becomes non-exponential and can be described by the distribution of rate constants. The results demonstrate shortening of the excited state lifetime in the presence of silver and broadening of the lifetime distribution caused by the nanoholes.

Toward the Development of an On-Chip Acoustic Focusing Fluorescence Lifetime Flow Cytometer

Conventional flow cytometry is a valuable quantitative tool. Flow cytometers reveal physical and biochemical information from cells at a high throughput, which is quite valuable for many biomedical, biological, and diagnostic research fields. Flow cytometers range in complexity and typically provide multiparametric data for the user at rates of up to 50,000 cells measured per second. Cytometry systems are configured such that fluorescence or scattered light signals are collected per-cell, and the integrated optical signal at a given wavelength range indicates a particular cellular feature such as phenotype or morphology. When the timing of the optical signal is measured, the cytometry system becomes “time-resolved.” Time-resolved flow cytometry (TRFC) instruments can detect fluorescence decay kinetics, and such measurements are consequential for Förster Resonance Energy Transfer (FRET) studies, multiplexing, and metabolic mapping, to name a few. TRFC systems capture fluorescence lifetimes at rates of thousands of cells per-second, however the approach is challenged at this throughput by terminal cellular velocities. High flow rates limit the total number of photons integrated per-cell, reducing the reliability of the average lifetime as a cytometric parameter. In this contribution, we examine an innovative approach to address this signal-to-noise issue. The technology merges time-resolved hardware with microfluidics and acoustics. We present an “acoustofluidic” time-resolved flow cytometer so that cellular velocities can be adjusted on the fly with a standing acoustic wave (SAW). Our work shows that acoustic control can be combined with time-resolved features to appropriately balance the throughput with the optical signals necessary for lifetime data.

Slr0320 Is Crucial for Optimal Function of Photosystem II during High Light Acclimation in Synechocystis sp. PCC 6803.

Upon exposure of photosynthetic organisms to high light (HL), several HL acclimation responses are triggered. Herein, we identified a novel gene, slr0320, critical for HL acclimation in Synechocystis sp. PCC 6803. The growth rate of the Δslr0320 mutant was similar to wild type (WT) under normal light (NL) but severely declined under HL. Net photosynthesis of the mutant was lower under HL, but maximum photosystem II (PSII) activity was higher under NL and HL. Immunodetection revealed the accumulation and assembly of PSII were similar between WT and the mutant. Chlorophyll fluorescence traces showed the stable fluorescence of the mutant under light was much higher. Kinetics of single flash-induced chlorophyll fluorescence increase and decay revealed the slower electron transfer from QA to QB in the mutant. These data indicate that, in the Δslr0320 mutant, the number of functional PSIIs was comparable to WT even under HL but the electron transfer between QA and QB was inefficient. Quantitative proteomics and real-time PCR revealed that expression profiles of psbL, psbH and psbI were significantly altered in the Δslr0320 mutant. Thus, Slr0320 protein plays critical roles in optimizing PSII activity during HL acclimation and is essential for PSII electron transfer from QA to QB.

Probing the structural features and the micro-heterogeneity of various deep eutectic solvents and their water dilutions by the photophysical behaviour of two fluorophores

The structural features of a series of diverse Deep Eutectic Solvents (DESs) have been investigated and characterized by means of two fluorescent probes. The spectral and photophysical properties of the latter are strictly dependent on the experienced environment, so that they can provide insights into the polarity, viscosity, hydrogen-bond network, and micro-heterogeneity of the various DESs.In fact, the investigated DESs exhibit a variety of properties with regards to their hydrophilicity, acidity, and hydrogen-bond ability, and these details were deeply probed by the two fluorescent molecules. The effect of the addition of water, which is a key strategy for tuning the properties of these structured systems, was also tested. In particular, the excited state dynamics of the probes, measured by femtosecond-resolved transient absorption, proved instrumental in understanding the changes in the structural properties of the DESs, namely reduced viscosity and enhanced heterogeneity, as the water percentage increases. Differences between the various DESs in terms of both local microheterogeneity and bulk viscosity also emerged from the peculiar multi-exponential solvation dynamics undergone by the excited states of the probes.The appraisal of a different local heterogeneity, depending on either the nature of the DES or the amount of added water, was also supported by the maximum entropy method (MEM) applied to the fluorescence decay kinetics, which gave clear indications about the different micro-heterogeneity of the DES systems.

A single host phosphor Ca3(SiO3)3:Eu2+, Mn2+ with good monodispersity for phosphor-converted white LEDs

Abstract A single host phosphor Ca3(SiO3)3:Eu2+, Mn2+ (CSO: Eu2+, Mn2+) with good monodispersity has been synthesized by high temperature solid state reaction combined with precursor, using porous silica as template and raw material. The phase, size and surface morphology of the phosphor were measured by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Luminescence properties were characterized by photoluminescence excitation (PLE) and emission spectra from room temperature to 480 K. CSO: Eu2+ has strong excitation band in UV region and emits blue light. To compensate for the lack of orange-red emission region and avoid using multiple phosphors, Mn2+ ions are introduced into the Ca2+ sites. The remarkable enhancements of the orange band attributed to an efficient nonradioactive energy transfer from Eu2+ to Mn2+, which has been testified by the photoluminescence spectra and fluorescence decay dynamics. The emitting light of Eu2+, Mn2+ co-doped CSO could be modulated from cool white to warm white by changing the molar ratio of Eu2+ and Mn2+.