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During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing). [ABSTRACT FROM AUTHOR]

Heat treatment of milk and dairy products are indispensable for the dairy industry. This thermal processing intends to extend shelf life, improve quality of the milk and minimize the health risks associated with milk and dairy products. The use of time-resolved fluorescence techniques to identify conformation and structure changes ok milk fat and proteins could help understand the temperature effects in bovine milk. This study aimed to use fluorescence lifetimes to evaluate the effects of heating fresh cow milk up to 85 °C. We observed different tendencies for fluorescence lifetimes submitted to different heating temperatures. The longer lifetime values decreased for temperatures higher than room temperature until it reached a minimum value near 40 °C and it slowly increased again for temperatures higher than 40 °C, indicating two distinct processes. These results indicate that time-resolved fluorescence can assist on the analysis of heating effects in fluid milk. Unlabelled Image • We propose a novel method for evaluation of effects on milk mild heating. • Fluorescence lifetime decreases linearly from room temperature until 40 °C. • Fluorescence lifetime increases slowly for temperatures higher than 40 °C. • Time-resolved fluorescence can assist on the analysis of mild heating effects on milk. • Vitamin A's fluorescence lifetime is correlated to milk's mild heating. [ABSTRACT FROM AUTHOR]

Time-resolved optical Kerr fluorescence system was used to investigate time-resolved red-shift of coumarin 153 in different solvent mixtures. The mixtures included four mole fractions of phenol-cyclohexane solvents (0, 0.013, 0.08, and 0.3), and anisole-cyclohexane solvents with the mole fraction of 0.3. The measured time-resolved fluorescence showed that, in the solvent mixtures containing phenol, the time-dependent frequency shift accelerated with the increase in the mole fraction of phenol-cyclohexane mixtures. However, the time-dependent red-shift in the fluorescence was not observed in the anisole-cyclohexane mixture, the solvent polarity could not influence the spectral Stokes shift compared with phenol. The results indicated that coumarin 153 formed an excited hydrogen bond with phenol, and the excited hydrogen bond was strengthened with an increase in the mole fraction of phenol. And, these processes also suggested that the increase in the phenol ratio improves a large number of hydrogen bond formed between phenol and carbonyl group of coumarin 153, the charge distribution will be faster towards lower the free energy of the system due to the stronger dipole moment. Therefore, the corresponding solvation response in phenol-cyclohexane mixtures with higher mole fractions decays very rapidly. Unlabelled Image • Time-resolved fluorescence spectra of C153-phenol and C153-anisole were measured. • Time-dependent red-shift becomes more obvious as phenol increases. • The excited hydrogen bond is strengthened with an increase in the phenol. • Dynamic process is faster in larger mole fraction of phenol-cyclohexane. • For anisole-cyclohexane, the time-dependent red-shift was not observed. [ABSTRACT FROM AUTHOR]

This paper presents a new strategy for qualitative identification of scopoletin and scopolin in Erycibe obtusifolia Benth using time-resolved (lifetimes) fluorescence and quantitative analysis with chemometrics-assisted excitation–emission matrix (EEM) fluorescence. Due to the significant spectral overlapping among analytes and interference, the use of the more selective time-resolved fluorescence is proposed for qualitative identification in quality control of traditional Chinese medicine (TCM) for the first time. Using the strategy of combining EEM fluorescence with second-order calibration method, i.e. parallel factor analysis (PARAFAC), the simultaneous quantification of scopoletin and scopolin in the complex system of Erycibe obtusifolia Benth was achieved successfully. The predicted concentrations were compared with the values obtained using high performance liquid chromatography-coupled to fluorimetric detector (HPLC-FLD), and no significant differences between them were observed. Therefore, the proposed methods using time-resolved fluorescence for qualitative analysis and EEMs coupled with second-order calibration for quantitative analysis in TCM are comparable and provide a suitable alternative to the chromatography-based method. Unlabelled Image • The use of the more selective time-resolved fluorescence is proposed for qualitative analysis in quality control of TCM. • Simultaneous quantification of scopoletin and scopolin was achieved successfully by EEMs combined with chemometrics. • PARAFAC was used to analyze EEMs. • The results by the proposed method were validated by HPLC-FLD. • The proposed strategy provides a simple and suitable alternative to the HPLC method. [ABSTRACT FROM AUTHOR]

Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna. • Photosystem I and Photosystem II work in series to drive photosynthesis in plants. • State transition balances the excitation pressure on the two photosystems. • The change in photosystem I antenna size during state transitions is quantified. • Light-harvesting complex II is an antenna of photosystem I in state 1 and state 2. [ABSTRACT FROM AUTHOR]

Time-resolved fluorescence (TRF) analysis is considered to be among the primary research tools in biochemistry and biophysics. One application of this method is the investigation of biomolecular interactions with promising applications for biosensing. For the latter context, time-correlated single photon counting (TCSPC) is the most sensitive, hence preferred implementation of TRF. However, high throughput applications are presently limited by the maximum achievable photon acquisition rate, and even more by the data processing rate. The latter rate is actually limited by the computational complexity to estimate accurately the fluorescence lifetime from TCSPC data. Here we propose a solution that would enable the implementation of TRF detection for fluorescence-activated droplet sorting (FADS), a particularly high throughput, microfluidic-based technology that can be used for drug discovery and thus help finding new cures for diseases. Most fluorescence lifetime algorithms require a large number of detected photons for an accurate lifetime computation. This paper presents an implementation based on a maximum likelihood estimator (MLE), enabling high precision estimation with a limited number of detected photons, significantly reducing the total measurement time. This speedup rapidly increases the input data rate. As a result, off-the-shelf embedded products cannot handle the data rates produced by current TCSPC units that are used to measure the fluorescence. Therefore, a configurable real-time capable hardware architecture is implemented on a field-programmable gate array (FPGA) that can handle the data rates of future TCSPC units, rendering high throughput droplet sorting with microfluidics possible. The presented hardware architecture is validated with experimental input data and produces high precision results. [ABSTRACT FROM AUTHOR]

Relaxation dynamics of excited state intramolecular proton transfer (ESIPT) probe 3‐formyl‐4‐hydroxy benzoic acid (FHBA) was studied by steady state, nanosecond time‐resolved fluorescence (TRF) and femtosecond transient absorption (TA) experiments in combination with adequate level of quantum chemical calculations. Global analysis of the transient data with target model reveals that enol conformers of both the neutral FHBA (I) and proton dissociated mono‐anionic species (III) undergo ESIPT in picosecond time scale (τESIPT ≈1.2 ps) towards the formation of corresponding vibrationally hot keto structure. Rapid intramolecular vibrational cooling (τ≈20 ps) follows singlet deactivation of the corresponding conformers with fluorescence lifetime (τfluo) of ∼5.1 and 2.0 ns, respectively as confirmed from stimulated emission (SE) and/or time resolved fluorescence experiments. Selective excitation of the di‐anionic structure (II) at 400 nm follows relatively simple excited state non‐ESIPT decay dynamics having fluorescence emission at 500 nm with τfluo ≈ 1.2 ns. Complete optimization of individual structures and construction of ESIPT profile was done by HF(CIS) as well as (TD)DFT calculation using 6–311++G(d,p) basis set and found to be consistent with the experimental observations. [ABSTRACT FROM AUTHOR]

The photoexcited processes in two compounds belonging to the salicylidene-p-X-anilines family were investigated by time resolved absorption and emission spectroscopy in the femto-to-millisecond time domain. The studies were conducted in two solutions of different polarity and were supplemented by investigations in solid state (microcrystalline powder). In the polar solvent, both compounds share similar fluorescence properties. In the non-polar solvent, one of the compounds exhibits only the tautomeric emission, while for the other compound, the fluorescence is almost quenched, but still visible as a co-existence of the normal and tautomeric emissions. These different emission characteristics in a non-polar solvent were correlated with the aggregate formation. From femto– to miliseconds, various deactivation channels and conformers of the known tautomeric forms have been evidenced altogether with an additional fluorescence relaxation appearing only in molecular aggregates. A complete and detailed image of the photochromic cycles emerges by correlating structural dynamics with the experimentally observed kinetic and spectroscopic parameters over 10 orders of magnitude time-scale. • The photophysical properties two Schiff bases have been studied by time resolved spectroscopy. • The photochromic cycles and the de-activation mechanism of excited states were drawn. • An additional fluorescence relaxation appears only in molecular aggregates. • Two simultaneous processes take place in excited state: E -enol → Z -enol isomerization and the very fast proton transfer. [ABSTRACT FROM AUTHOR]

The quenching of chlorophyll-a fluorescence was investigated in plant light-harvesting complex II (LHCII) embedded in reconstituted membranes containing thylakoid lipids and a lipid label. The proteoliposomes were further separated by density and the protein and lipid contents of the fractions were quantified spectrophotometrically, allowing tighter control over the L/P ratios in a wide range of values. Using time-resolved fluorescence, we found a strong correlation between the fluorescence quenching and the L/P ratio, in line with other studies reporting progressive quenching at low L/P ratios, presumably triggered by self-clustering in the membrane. The average fluorescence lifetimes decreased to 0.3 ns at L/P ratios below 50:1; these values are comparable to the quenching observed in plants under excess-light conditions and are accompanied by a similar far-red fluorescence signature. It is hypothesized that plants can exploit the intrinsic quenching propensity of LHCII-only membrane domains to safely store extra antenna units. [ABSTRACT FROM AUTHOR]

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Graphical abstract Highlights • Photocatalytic oxidation of phenolic pollutants is achieved using NMQ+. • Photocatalytic oxidation of phenolic pollutants is achieved by Mes-Acr-Me+. • Pollutants quench singlet excited state of the photocatalysts. • Kinetic rate constants (108–1010 M-1s-1) indicate Type I photodegradation. Abstract The photodegradation of a mixture of phenolic pollutants including: phenol (P), orto- phenylphenol (OPP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) was accomplished using two organic cationic photocatalysts, namely N -methylquinolinium (NMQ+) and 9-mesityl-10-methylacridinium (Mes-Acr-Me+) salts, due to their singular photophysical and redox properties. On one hand, NMQ+ exhibits more energetic excited states and accordingly more favorable redox potentials than Mes-Acr-Me+; on the other hand, NMQ+ absorption reaches only up to 380 nm, while Mes-Acr-Me+ extends in the visible up to 480 nm. Evaluation of the efficiency of both photocatalysts, revealed that the highest level of photodegradation was achieved when they were employed at 20% mol. Specifically, with NMQ+, removal of the pollutants was completed within 24 h of irradiation. Even more, irradiation time could be shortened from 24 to 8 h, since high levels of removal were already achieved (93%, 100%, 100% and 82% for P, OPP, TCP and PCP, respectively). Albeit, Mes-Acr-Me+ was not as effective, and best results were obtained using 20% mol upon 24 h of irradiation. Under these conditions, removal of PCP was 80%, while TCP was 40%, OPP 30% and P resulted in the most recalcitrant contaminant with only 10% of removal. Next, NMQ+ and Mes-Acr-Me+ were separately supported onto Zeolite Y, an inert inorganic support (Y-NMQ+ and Y-Mes-Acr-Me+), and elemental analyses revealed a loading of ca. 13% and 15% weight for NMQ+ and Mes-Acr-Me+, respectively. Upon heterogenization, in the case of Y-NMQ+, the extent of removal was lower than the one achieved in the homogeneous photodegradations. On the contrary, performance of Y-Mes-Acr-Me+ improved, because of its enhanced photostability; thus, upon 46 h irradiation, 98%, 80%, 40% and 26% for PCP, TCP, OPP and P, respectively, was achieved. Moreover, their efficiency was maintained upon second use. Steady-state and time-resolved fluorescence quenching revealed that every pollutant was able to quench the singlet excited state of both 1(NMQ+)* and 1(Mes-Acr-Me+)*, with kinetic rate constants in the order of the diffusion limit. Thus, Type I photooxidation happening through the singlet excited state of either photocatalyst was the main operating process in the photodegradation of the studied pollutants. [ABSTRACT FROM AUTHOR]

Abstract Monomeric zinc phthalocyanine has been studied as a promising active photosensitizer in photodynamic therapy against cancer, in which its aggregate form is non-active. This paper aimed to describe the monomer/aggregates equilibrium of zinc phthalocyanine in binary water/DMSO mixtures. To reach this aim theoretical calculation, electronic absorption, static and time-resolved fluorescence, and resonance light scattering was used. Zinc phthalocyanine shows a complex water dependence behavior in the mixture. At least three distinct steps were observed: (i) until 30% water zinc phthalocyanine is essentially in the monomeric form, changing to (ii) small slipped cofacial-aggregates around 30% to 40% water and finally to (iii) a staircase arrangement of large aggregates at higher water percent. The staircase arrangement is driven by the intermolecular coordination between the pyrrolic nitrogen lone-pairs and the central metal zinc. The water-Zn coordination governs the fluorescence quenching by a static mechanism. These results have direct relevance in the better understanding on the behavior of zinc phthalocyanine in vivo and when incorporated in drug delivery systems for clinical applications in photodynamic therapy. Graphical abstract Unlabelled Image Highlights • Monomer/aggregate equilibrium of zinc phthalocyanine at water/DMSO mixtures were studied. • ZnPc aggregates are driven by the intermolecular coordination between the pyrrolic nitrogen lone pairs and the central zinc. • ZnPc is in the monomeric form below 30% water, changing to small slipped cofacial aggregates at 30–40% water. • Staircase arrangement of large ZnPc aggregates at >40% water are present. • The findings may be useful to better understand ZnPc behavior in other systems, such as cancer cells for photodynamic action. [ABSTRACT FROM AUTHOR]

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation. [ABSTRACT FROM AUTHOR]

Abstract We report photophysical properties of 1,2-dihydroxyanthraquinone (alizarin) confined in methanol-in-oil reverse micelles by steady-state and time-resolved fluorescence spectroscopy. The formation of reverse micelles and confinement of alizarin molecules inside the reverse micelles were confirmed by Raman spectroscopy and fluorescence anisotropy measurements. The emission of alizarin centered at 585 nm showed 2.5–3.0 times increase in intensity in small reverse micelles, and the lifetime was also largely increased compared to those of bulk solution. We conclude that alizarin confined in the core of small reverse micelles has a strong interaction with the sulfonate head group of the surfactant Aerosol OT, and that the emission band at 585 nm originates from the alizarin with the proton partly deprotonated and trapped near the anionic surfactant head groups. Graphical abstract Unlabelled Image Highlights • Alizarin (1,2-dihydroxyanthraquinone) exists inside the core of methanol/AOT/isooctane reverse micelles. • Alizarin deprotonates partially in the reverse micelles and the excited state lifetimes are strongly increased. • The emission of alizarin in the small reverse micelles increases strongly with a new emission band at 585 nm. • Alizarin is considered as trapped between the negatively charged head groups of the surfactant AOT. [ABSTRACT FROM AUTHOR]

Photochromic ring closure reaction dynamics of 1,2‐bis(2‐methylbenzo[b]thiophene‐3‐yl)hexafluoro cyclopentene is investigated by means of time‐resolved fluorescence spectra. Compared with a single wavelength probe, direct measurement of the emission spectra during the reaction provides unambiguous and straightforward picture for the ring closure reaction pathway. We observe two different emission bands in the fluorescence of the open ring isomer. From the global analysis for the spectra, we obtain <1 ps time constant for the ring closure reaction in polar solvent. Moreover, the ring closure reaction of the compound takes place entirely on the ultrafast timescale. The unreactive parallel conformer decays to the ground state by 150 ps time constant. [ABSTRACT FROM AUTHOR]

Time-resolved fluorescence measurement is extraordinarily powerful in the analysis of substances due to its effectiveness in eliminating measurement artifacts. Some fluorescence measurements are still conducted on CMOS chips with the decay times determined after reading the data off the chip and fitting the fluorescence decay histogram. We present a novel approach in which an analog CMOS chip divides the fluorescence decay time into slices and classifies the photons according to their arrival times at a CMOS SPAD sensor. The chip was fabricated in a 1P6M 0.18 μm HV-CMOS process. The slice timings can be tailored from 168 ps to 4.9 ns, covering most fluorescence decay times. 9 timing windows are generated per pixel that count up to 13 b each, with a resolution of 0.16 mV/photon, for a maximum output voltage of 1.3 V, in an area of 150 μm × 50 μm. Here, we report on the first practical application of this circuit, which integrates an array of 5 pixels in a single chip and has an excitation light and a microfluidic chip of up to 3 channels. This system could determine the decay time of quantum dots in 20 nl of solution. Thus, this paper could help in the development of a point-of-care device based on time-resolved fluorescence. [ABSTRACT FROM AUTHOR]

Abstract The host-guest interaction of a 7-hydroxycoumarin (3-[2-(diethylamino)ethyl]-7-hydroxy-4-methylcoumarin, 3ED4MU) with cucurbit[7]uril, CB7, was investigated in aqueous solution. From the steady-state fluorescence Job plot and 1H NMR spectroscopy the formation of a 1:1 inclusion complex, with a binding constant of 1.75 × 105 M−1 was obtained. Time-resolved fluorescence data in water, DMSO and dioxane for 3ED4MU and for umbelliferone (UM , here taken has the model compound) show that the decays are double or triple-exponentials mirroring the presence of excited neutral (N*), anionic (A*), tautomeric (T*) and zwitterionic (T*′) species (the latter only present in 3ED4MU). In the time-resolved experiments, the interaction of 3ED4MU with CB7 is mirrored by an increase of the pre-exponential factor associated to the zwitterionic species (thus giving support for the stabilization of this species when interacting with CB7) and a dramatic change in the pK a * value (from ~0.5 to ~8), associated to a displacement of the water environment probed by 3ED4MU. Highlights • Interaction studies with a 4-methylumbelliferone derivative and cucurbit[7]uril (CB7) • 1:1 stoichiometry between the 4-methylumbelliferone derivative (3ED4MU) and CB7 • Binding constants of 1.75 × 105 M−1 (ground state) and 2.30 × 105 M−1 (excited state, ES) • 3ED4MU-CB7 complex leads to a dramatic change of the acid-base equilibria in the ES. [ABSTRACT FROM AUTHOR]

Graphical abstract The illustration depicts the favorable interaction initiated by introduction of a potential therapeutic bioorganic luminophore from the class of Azapodophyllotoxin into the sudlow site I of human serum albumin and its ability to regulate the innate rotamers of Trp-214 residue in Human Serum Albumin. Highlights • Adaptation of therapeutic luminophore Azapodophyllotoxin into sudlow site I of HSA. • Electrostatic & hydrophobic interaction are major factors into adapting HPFQ. • Minor perturbation occurs in secondary structure of malleable sudlow site I of HSA. • Molecular docking assists to establish the close proximity of HPFQ to Trp-214. • Exploration of FRET mechanism between HPFQ and Trp-214 of HSA. • HPFQ regulates the emissive population of three rotamers of Trp-214 in HSA. Abstract Human serum albumin is perceived to be the most abundant protein in human blood plasma and functions as a major carrier of different enzymes and drugs inside human body. The present article puts in an effort to demonstrate the attitude adopted by human serum albumin towards a potential therapeutic luminophore 4-(2-Hydroxyethyl)-10-phenyl-3,4,6,7,8,10-hexahydro-1H-cyclopenta[ g ]furo[3,4-b]quinoline-1-one (HPFQ). HPFQ is a prodigy from azapodophyllotoxin class of compounds, which have been synthesized from the perspective of improved bioactivity than its prologue podophyllotoxins. While, HPFQ has proved to be highly bioactive against most cancer cell lines with best GI 50 values of <0.1 µM for a major number of cell lines; it also showed terrific fluorescent properties throughout the polarity scale, worthy of a promising imaging agent. The binding mechanism of HPFQ with HSA has been established by combining in vitro spectroscopic techniques, in silico molecular docking and induced fit docking (IFD). The competitive site-binding studies demonstrated that the otherwise anion-receptor sudlow site I of HSA nurtures neutral HPFQ with prudent affinity (Binding constant, K b = 0.74 × 105 M−1). The time-resolve fluorescence studies reveal an appreciable reduction in HSA average radiative lifetime against an increase in HPFQ concentration and provided evidence for Forster's resonance energy transfer (FRET) being responsible for the dominant quenching mechanism, escorted by minor structural deformations in the backbone of protein structure. HPFQ institutes itself near Trp-214 within protein matrix, and subsequently the "hydrophobic amino acids" dominated cybotactic environment of Trp-214 experiences a reduction in the micropolarity. The allosteric modulation triggered by the stronger association of HPFQ with HSA leads towards minor deformation in secondary structure of protein. Sudlow site I of HSA proficiently embraces a favourable conformation like malleable dough to furnish space for arriving bioactive HPFQ molecule. HPFQ is also believed to administer the conformational regulation in HSA domain by affecting inter-conversion of HSA rotamers, which may prove to be an enlightening area to decode the preferable interaction between them. The juxtaposed spectroscopic research described herein is expected to embolden design of azapodophyllotoxin based anti-proliferative clinical agents for efficient in vivo bio-distribution employing HSA-centred drug delivery and administration systems. [ABSTRACT FROM AUTHOR]

Abstract The binding interactions between bovine serum albumin (BSA) and a large excess of uric acid (UA) have been analyzed in aqueous solution at pH = 7.4, using UV–vis absorption and steady-state, time-resolved, synchronous and three-dimensional fluorescence spectra techniques. The observed effects verify the formation of π-π stacked non-covalent and non-fluorescent complexes in the system BSA-UA. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as the measurements of fluorescence decay times and Förster resonance energy transfer parameters validate the mechanism of static quenching. The changes in the synchronous fluorescence spectra indicate that the BSA fluorescence quenching originates both from Trp and Tyr residues, but their involvement is not equivalent. The interaction with UA induces the diverse alterations of BSA tertiary structure: folding of the polypeptide chains around Trp residues and concurrently their extension around Tyr residues. The obtained outcomes suggest that the presence of the excessive amount of UA in blood could interfere with the physiological functions of serum albumin, especially in case of UA overproduction. Graphical abstract Unlabelled Image Highlights • The interaction between BSA and uric acid has been investigated. • Uric acid quenches the BSA fluorescence by static quenching process. • Binding, fluorescence quenching and bimolecular quenching constants were calculated. • FRET data, time-resolved, synchronous fluorescence prove stacking complexes formation. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Mercaptoacetic acid (MAA) is used to control the size and for surface functionalization of CuO QDs. • Photoresponsive azobenzene ligand, (E)-5-((4-chlorophenyl) diazenyl)–2-hydroxy benzaldehyde (CPDHB) is attached to QDs. • Photoinjected electron is transferred from the CB of CuO QDs to the LUMO of CPDHB. • The photoinduced charge/energy transfer in QDs-CPDHB system may lead to design fluorescent probes and data storage devices. Abstract A series of stable CuO quantum dots (QDs) in aqueous alkaline medium are prepared and protected against rapid oxidation by capping with mercaptoacetic acid (MAA). The MAA not only regulates the size of the CuO QDs but it also acts as pendent group for further functionalization of QDs. A specific concentration of MAA improves the crystallinity and increases the fluorescence lifetime by decreasing the density of surface trap states. The MAA capped CuO QDs are attached to (E)-5-((4-chlorophenyl) diazenyl)-2-hydroxybenzaldehyde (CPDHB) and upon UV irradiation at 300 nm the CPDHB experiences a reversible trans- to cis-isomerization. The absorption band of the cis-isomer of CPDHB overlaps with the fluorescing band of the CuO QDs, as a result the fluorescence of the QDs is strongly quenched. The photoluminescenc (PL) quenching is attributed due to the photoinjected electron transfer from the conduction band (CB) of the CuO QDs to the lowest unoccupied molecular orbital (LUMO) of the cis-isomer of CPDHB. Förster resonance energy transfer (FRET) is also observed but its contribution is minor. The fluorescence quenching of CuO QDs in QDs - CPDHB assembly upon ultra-violet excitation and reversible trans-cis interconversion of CPDHB may potentially suggests to design fluorescent probes for bioimaging and to develop data and energy storage devices. [ABSTRACT FROM AUTHOR]

beta-nicotinamide adenine dinucleotide (NADH)-alcohol dehydrogenase complex was compared to either UVA irradiation (364 nm; 50 mW cm-2; 0-60 min) or heat in order to investigate complex stability. Prior to irradiation, frequency-domain fluorescence lifetime measurements indicated the presence of two principal components having short (subnanosecond) and long (nanosecond) fluorescence lifetimes reflecting free and bound NADH respectively. UVA exposure resulted in decreased NADH fluorescence intensity concomitant with decreased absorption at 337 nm. However, UVA irradiation did not reduce the fractional contribution of the long-lived bound NADH. The photoinduced fluorescence decrease appeared to be caused by the formation of oxidized NAD+ and not on UVA-induced dissociation of the NADH-protein complex. Such dissociation, detected by a red-shifted fluorescence maximum and decreased fractional contribution of the nanosecond component, was observed when NADH-protein mixtures were heated.

beta-nicotinamide adenine dinucleotide (NADH)-alcohol dehydrogenase complex was compared to either UVA irradiation (364 nm; 50 mW cm-2; 0-60 min) or heat in order to investigate complex stability. Prior to irradiation, frequency-domain fluorescence lifetime measurements indicated the presence of two principal components having short (subnanosecond) and long (nanosecond) fluorescence lifetimes reflecting free and bound NADH respectively. UVA exposure resulted in decreased NADH fluorescence intensity concomitant with decreased absorption at 337 nm. However, UVA irradiation did not reduce the fractional contribution of the long-lived bound NADH. The photoinduced fluorescence decrease appeared to be caused by the formation of oxidized NAD+ and not on UVA-induced dissociation of the NADH-protein complex. Such dissociation, detected by a red-shifted fluorescence maximum and decreased fractional contribution of the nanosecond component, was observed when NADH-protein mixtures were heated.

• Curcumin is recognized for its potent antioxidant and anti-inflammatory properties. • β-lactoglobulin is a globular protein found in the milk of various mammalian species. • α-lactalbumin exhibit potential therapeutic benefits in various health conditions. Understanding the interaction between nano-curcumin (Nano Cur) and proteins is essential for elucidating the potential therapeutic applications of curcumin in nano formulations. In this study, we investigated the interactions of Nano Cur and two important milk proteins, β-lactoglobulin and α lactalbumin as binary and ternary systems, in molecular-level through the employment of various biophysical methods including various different spectroscopies, and molecular dynamic simulations. Fluorescence spectroscopy analysis revealed distinct changes in the fluorescence emission of both proteins upon binding to Nano Cur as an indication of protein-Nano Cur complexes formation. The non-linear Stern-Volmer plots of β-lactoglobulin-Nano Cur complex formation in the absence and presence of α-lactalbumin displayed two set of binding sites that referred to the dominance of two different interaction behaviors with two set of binding sites. Also, the comparison between K sv and K b values of β-lactoglobulin-Nano Cur in the absence and presence of α-lactalbumin with the temperatures revealed the influence of combined dynamic and static quenching. Circular dichroism measurements further provided insights into the potential enhancement of the secondary structure of proteins upon interaction with Nano Cur through the induced alterations. In order to access a deeper view on the dynamic behavior of protein-Nano Cur complexes, time-resolved fluorescence measurements were performed to reveal the kinetics of interaction process. Additionally, molecular dynamic simulations were employed to simulate the atomic-level interactions and explore the stability and conformational changes of protein-Nano Cur complexes over time. The results of these comprehensive biophysical investigations shed light on the binding characteristics, structural alterations, and dynamic behavior of Nano Cur upon interaction with β-lactoglobulin in the absence and presence of α-lactalbumin as the binary and ternary systems. This study reveals the Nano-Cur role in the protein–protein interaction behavior and provides valuable insights into the potential applications of α-lactalbumin in the design of functional food formulations and drug delivery systems. [ABSTRACT FROM AUTHOR]

Thin microcrystals of pyrene have been recrystallized by using a surfactant. With a size-range of several hundred nm to a few micrometers, the crystals have been characterized by using standard techniques of X-ray and Raman spectroscopy besides the atomic force and scanning electron microscopy. The fluorescence microscopy of single microcrystal shows fluorescence due to monomer as well as the red-shifted broad, structureless excimer-type emission. The observed decay profiles of the red-shifted emission reveal existence of dimer and higher aggregates in the crystalline state when excited at the red-edge of the absorption band. This is in contrast to the excimer emission observed at higher energy excitation range corresponding to the monomer absorption. Distinctly different nature of time-resolved decay profiles differentiates the two mechanisms. The observations have been used to propose existence of different sites of crystal packing under two different excitations (370 nm and 408 nm). The structural anisotropy of these crystals makes them useful as a probe in crystal structure based molecular and biological sensing. • Pyrene microcrystals have been synthesized in the presence of a surfactant to control their thickness. • The excimer, dimer and higher oligomers formation are distinguished using time-resolved studies. • There is no evidence of excimer formation at the red edge excitation. [ABSTRACT FROM AUTHOR]

Interaction of an antibody with its FcγR plays an important role in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). Nowadays altered ADCC activity of an antibody can be achieved by utilizing an effective glyco-engineering strategy, which often involves changes of sugar moieties in Fc part of the antibody, thereby affecting its receptor binding with effector cells. We aimed to construct a cell-based time-resolved fluorescence (TRF) assay for the evaluation of ADCC activity triggered by the antibody drug Trastuzumab (anti-HER2) and T-DM1. The assay was initiated by incubating 2,2′:6′,2 "-Terpyridine-6,6"-dicarboxylic acid (TDA)-labeled target SK-BR3 cells with the testing antibodies and engineered NK-92 effector cells. After incubation, the target cells were lysed to detect TDA released into the supernatant. Together with added Eu, the TDA in the supernatant formed a stable chelate of EuTDA with high-intensity fluorescence. The ADCC activity was then determined by measuring the fluorescence of EuTDA. Consequently, the method demonstrated good accuracy, precision, linearity, and specificity over methodological assessment and compared well with the Luciferase release assay in terms of the agreement of the achieved results. Using the developed assay, we evaluated the ADCC activity of two glyco-engineered anti-HER-2 antibody-drug conjugates (ADCs) and the results showed that antibody Fc glycosylation modifications influenced antibody ADCC activity to varying degrees. In conclusion, the present assay is able to accurately assess the ADCC activity induced by Trastuzumab (anti-HER2) and T-DM1, and a similar methodology can be applied to other therapeutic antibodies during drug development to help screen for antibodies with desirable ADCC activity. • A cell-based TRF assay for analysis of ADCC activity was developed and evaluated. • Comparison between the cell-based TRF assay and the Luciferase release assay was conducted. • The ADCC activity of Trastuzumab and three anti-HER2 antibody-drug conjugates were investigated. [ABSTRACT FROM AUTHOR]

• Steroid hormone (SH) removal in a TiO 2 coated PES membrane was quantified • Organic matter can assume role of enhancer or inhibitor to SH degradation • As a photosensitizer, humic acid at 5 mgC/L enhanced SH removal from 47 to 72% • Superoxide radical was identified as a predominant species for SH degradation • Tannic acid inhibited the SH removal by screening light and scavenging reactions Water treatment in photocatalytic membrane reactors (PMR) holds great promise for removing micropollutants from aquatic environments. Organic matter (OM) that is present in any water matrix may significantly interfere with the degradation of steroid hormone (SH) micropollutants in PMRs. In this study, the interference of various OM types, humic acid (HA), Australian natural organic matter (AUS), worm farm extract (WF), tannic acid (TA), and gallic acid (GA) with the SH degradation at its environmentally relevant concentration (100 ng/L) in a flow-through PMR equipped with a polyethersulphone-titanium dioxide (PES-TiO 2) membrane operated under UV light (365 nm) was investigated. Results of this study showed that OM effects are complex and depend on OM type and concentration. The removal of β-estradiol (E2) was enhanced by HA at its levels below 5 mgC/L while the enhancement was abated at higher HA concentrations. The E2 removal was inhibited by TA, and GA, while no significant interference observed for AUS, and WF. The data demonstrated diverse roles of OM that acts in PMRs as a light screening agent, a photoreactive species scavenger, an adsorption alteration trigger, and a photosensitizer. The time-resolved fluorescence measurement showed that HA, acting as a photosensitizer, promoted the sensitization of TiO 2 by absorbing light energy and transferring energy/electron to the TiO 2 substrate. This pathway dominated the mechanism of the enhanced E2 degradation by HA. The favorable effect of HA was augmented as increasing the light intensity from 0.5 to 10 mW/cm2 and was weakened at higher light intensities due to the increased scavenging reactions and the limited amount of HA. This work clarifies the underlying mechanism of the OM interference on photocatalytic degradation of E2 by the PES-TiO 2 PMR. [Display omitted] [ABSTRACT FROM AUTHOR]

Band-gap engineering of graphene-based nanomaterials has a huge influence on their optical and structural properties. Our contribution reports the successful gram-scale synthesis of highly chlorinated graphene quantum dots (Cl-GQDs) via a two-step preparation method. X-ray photoelectron spectroscopy (XPS) proved the successful doping of chlorine atoms into the as-prepared graphene oxide quantum dots (O-GQDs). Time-resolved fluorescence revealed a high decrease in the relaxation lifetime from 690 ps for O-GQDs to 5.3–4 ps for Cl-GQDs. Transmission electron microscopy (TEM) confirms that all nanoparticles are on the quantum scale (4–6 nm). The large blueshift in photoluminescence (PL) 150 nm from the original O-GQD (440 → 290 nm) supports the chlorination effect which enables tuning the band gap of O-GQD. [Display omitted] [ABSTRACT FROM AUTHOR]

By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.

By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.

CdSe/CdS quantum dots (QDs) were seeded into Jurkat cells using polyethylene glycol (PEG-1500) at different treatment times. Fluorescence microscopy images show that some QDs stick to the surface of the cells, while others appeared to be inside the cells. As it is difficult to ascertain whether the QDs are indeed inside the cells or just behind the cells, additional spectroscopic studies were performed. Photoluminescence spectra show that the fluorescence intensities of the CdSe/CdS QDs are different between samples at different treatment times. Interestingly, the fluorescence lifetimes are also different. This confirms the interaction between the CdSe/CdS QDs and the intracellular media and that the QDs were successfully seeded into the cells.

Nanomaterials have been widely used in biomedical sciences; however, the mechanism of interaction between nanoparticles and biomolecules is still not fully understood. In the present study, we report the interaction mechanism between differently sized Ag nanoparticles and the improved light-oxygen-voltage (iLOV) protein. The steady-state and time-resolved fluorescence results demonstrated that the fluorescence intensity and lifetime of the iLOV protein decreased upon its adsorption onto Ag nanoparticles, and this decrease was dependent upon nanoparticle size. Further, we showed that the decrease of fluorescence intensity and lifetime arose from electron transfer between iLOV and Ag nanoparticles. Moreover, through point mutation and controlled experimentation, we demonstrated for the first time that electron transfer between iLOV and Ag nanoparticles is mediated by the tryptophan residue in the iLOV protein. These results are of great importance in revealing the function of iLOV protein as it applies to biomolecular sensors, the field of nano-photonics, and the interaction mechanism between the protein and nanoparticles.

Fluorescence spectroscopy was used to study a solution comprised of coumarin 153 (C153)+ trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14]+ [Tf2N]−)+ supercritical CO2 (scCO2). We compare the spectroscopy of C153 in neat scCO2 to that of C153/scCO2 with the addition of ionic liquid (IL). Excitation and emission peak frequencies of C153 in scCO2 and in IL/scCO2 diverged at reduced densities (ρr = ρ/ρc) below the CO2 critical density. At low fluid density, spectral changes in the IL/scCO2 solutions showed evidence that C153 experiences a very different microenvironment—one that is unlike neat scCO2. The data show that the presence of IL clearly influences the C153 excitation and emission profiles. Excitation was broadened and red shifted by >2000 cm−1 and the presence of an additional low-energy emission component that was red shifted by ~3000 cm−1 was clearly visible and not observed in neat scCO2. The solution heterogeneity was controlled by changing the scCO2 density and at high fluid density, both the excitation and emission spectra were more similar to those in neat scCO2. Steady-state anisotropy also showed that at low fluid density, the C153 emission was significantly polarized. Aggregation of C153 has been reported in the literature and this led us to hypothesize the possibility that C153 dimer (aggregation) formation may be occurring in scCO2. Another possible explanation is that dye–IL aggregates may dissolve into the scCO2 phase due to C153 acting as a “co-solvent” for the IL. Time-resolved intensity decay measurements yielded only slightly non-exponential decays with accompanying time constants of ~3–4 ns that were significantly shorter than the 5–6 ns time constants in neat scCO2, which are suggestive of C153–IL interactions. However, these data did not conclusively support dimer formation. Pre-exponential factors of the time constants showed that almost all of the emission was due to monomeric C153.

The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microheterogeneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 7224-7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state.

The photophysical mechanisms which determine the spectral properties and decay rates of 4',6-diamidine-2-phenylindole (DAPI) in solution and in association with nucleic acids have not yet been fully elucidated. We have performed steady-state and time-resolved fluorescence experiments on DAPI in a wide pH range to investigate the hypothesis that different ground-state conformations are responsible for the photophysical properties of the probe. Several excited-state mechanisms are investigated and it is concluded that among the proposed models, the hypothesis of ground-state heterogeneity with rapid interconversion among conformations is the only one consistent with the experiments in the entire pH range investigated.

Kidney disorders are a rising global health issue, necessitating early diagnosis for effective treatment. Creatinine, a metabolic waste product from muscles, serves as an ideal biomarker for kidney damage. The existing optical methods for creatinine detection often involve labor-intensive synthesis processes and present challenges with the aqueous solubility and sensitivity to experimental variations. In this study, we introduce a straightforward fluorescence "turn-on" ratiometric sensor system for creatinine detection in aqueous media with a limit of detection of 0.5 μM. The sensor is based on sulfated-β-cyclodextrin (SCD)-templated H-aggregate of a commercially available, ultrafast rotor dye thioflavin-T (ThT). The Al 3+ ion-induced dissociation of ThT-SCD aggregates, followed by reassociation upon creatinine addition, generates a detectable signal. The modulation of monomer/aggregate equilibrium due to the disassembly/reassembly of the ThT-SCD system under Al 3+ /creatinine influence serves as the optimal strategy for ratiometric creatinine detection in aqueous media. Our sensor framework offers several advantages: utilization of the readily available dye ThT, which eliminates the need for a laborious synthesis of custom fluorescent probes; ratiometric sensing, which improves quantitative analysis accuracy; and compatibility with complex aqueous media. The sensor's practical utility has been successfully demonstrated in artificial urine samples. In summary, our sensor system represents a significant advancement in the rapid, selective, and sensitive detection of the clinically crucial bioanalyte creatinine, offering potential benefits for the early diagnosis and management of kidney disorders.

Abstract The objective of this work was to evaluate the efficacy of time-resolved and stationary fluorescence spectroscopic techniques in the detection of adulterations in Whey Protein Concentrate (WPC) powder, used in nutritional supplements. Adulterations were performed by the individual addition of caffeine, creatine and lactose in WPC in different levels (10%, 20% and 30% w/w). Samples were submitted to 275 nm excitation wavelength. Time-resolved fluorescence revealed that the adulterations changed the lifetimes of the tri-exponential decay curves obtained at 335 nm. The 30% adulterations demonstrated higher values of the mean emission lifetime, with caffeine treatment statistically significant. The fluorescence spectra showed an emission peak at approximately 335 nm with a broadband between 325 and 430 nm in the caffeine treatment. Euclidean distance analysis indicated the creatine and lactose treatments (10, 20 and 30% w/w) as similar to control WPC, while caffeine 20% and 30% treatments not. Hierarchical Cluster Analysis could differentiate all caffeine treatments from the other samples. Principal Component Analysis confirmed the differentiation of all adulterants from the control WPC. In general, the application of time-resolved fluorescence and stationary fluorescence spectroscopy techniques allowed the characterization and observation of differences among the samples, when allied to statistical tools. Highlights • Adulterations in formulations of Whey Protein Concentrate powder were evaluated. • Time-resolved fluorescence raised the mean lifetime decay in 30% caffeine treatment. • Fluorescence emission maps showed visual differences among the samples. • HCA separated caffeine treatments from the others. • PCA discriminated all the samples. [ABSTRACT FROM AUTHOR]

A novel copolymer poly(thiophene-2,5-diyl-2,5-di-n-octyloxycarbonyl-1,4-phenylene), denoted as P33, is introduced as potential material for photovoltaics, polymer light-emitting diodes, and/or organic transistors. P33 dissolved in chloroform is investigated by steady-state absorption, linear/non-linear fluorescence spectroscopies and time-resolved fluorescence spectroscopy. Molar extinction coefficient, fluorescence quantum yield, and singlet fluorescence lifetime of P33 are determined to be 18,315 M−1 cm−1, 0.4, and 810 ps, respectively. The P33 fluorescence fast components of decay times are 1.2 ps, 2.0 ps, and 0.5 ps for increasing wavelengths of 480 nm, 500 nm, and 520 nm, respectively. The fast component is attributed to a transport of nearly instantaneously formed excitons to localized states known as downhill energy transfer. Additionally multi-photon excited fluorescence is observed for pumping with wavelengths of 800 nm and 1200 nm. Two-photon absorption cross-section is determined to be 6.9 GM. These spectroscopic studies provide basic fluorescence characteristics of the novel thiophene copolymer P33. [ABSTRACT FROM AUTHOR]

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation. [ABSTRACT FROM AUTHOR]

Recently, we proposed a simple conceptual fluctuating antenna model (FAM), describing excitation diffusion and trapping in a continuous medium, where variations of the excitation transfer pathways are taken into account by the introduced fractional space dimension. Since then, this model has been successfully applied to simulate multi-exponential excitation quenching kinetics in a series of plant photosynthetic systems, purified from the thylakoid membranes, without invoking a radical pair state in the reaction centre. Here, we overview this model and its parameters obtained for various systems, and extend the area of its applications to several pigment-protein supercomplexes containing the photosystem I (PSI). We show that while the diffusion in the PSI core is virtually three-dimensional, the PSI core aggregates interconnected with other light-harvesting complexes (LHCI and/or LHCII) are characterized by a substantially reduced dimension, which indicates a smaller number of energy transfer links from LHCI to the PSI core. We also suggest that in vivo both PSI and PSII antennae are substantially larger than those observed in the isolated systems: PSII antenna contains in total about 6 LHCII trimers while PSI is aggregated with at least one LHCII trimer. The obtained results show that FAM can be a very useful tool to follow photosynthetic apparatus transformations during short- and long-term adaptation to varying light, monitored by kinetic fluorescence spectroscopy. [ABSTRACT FROM AUTHOR]

Non-photochemical quenching (NPQ) is responsible for the protection of the photosynthetic apparatus of plants from photodamage at high-light conditions. It is commonly agreed that NPQ takes place in the major light-harvesting complexes (LHCII), however, its exact mechanisms are still under debate. Valuable information about its molecular nature can be provided by measuring time-resolved fluorescence (TRF) spectra of LHCII complexes and their aggregates. Previously [Chmeliov et al., Nat. Plants 2, 16045 (2016)], we analysed the corresponding TRF spectra using the multivariate curve resolution method and proposed a three-state model to describe the spectroscopic data. Usually, such data is described in terms of global analysis resulting in decay or evolution-associated spectra. In this work, we apply such analysis to the TRF data of LHCII aggregates and show that, although mathematically feasible, it cannot be directly related to the physical kinetic model. Nevertheless, a careful examination supplemented with additional spectroscopic information still results in the same three-state model proposed before. [ABSTRACT FROM AUTHOR]

Low-temperature fluorescence measurements are frequently used in photosynthesis research to assess photosynthetic processes. Upon illumination of photosystem II (PSII) frozen to 77 K, fluorescence quenching is observed. In this work, we studied the light-induced quenching in intact cells of Chlamydomonas reinhardtii at 77 K using time-resolved fluorescence spectroscopy with a streak camera setup. In agreement with previous studies, global analysis of the data shows that prolonged illumination of the sample affects the nanosecond decay component of the PSII emission. Using target analysis, we resolved the quenching on the PSII-684 compartment which describes bulk chlorophyll molecules of the PSII core antenna. Further, we quantified the quenching rate constant and observed that as the illumination proceeds the accumulation of the quencher leads to a speed up of the fluorescence decay of the PSII-684 compartment as the decay rate constant increases from about 3 to 4 ns− 1. The quenching on PSII-684 leads to indirect quenching of the compartments PSII-690 and PSII-695 which represent the red chlorophyll of the PSII core. These results explain past and current observations of light-induced quenching in 77 K steady-state and time-resolved fluorescence spectra. [ABSTRACT FROM AUTHOR]

Advanced oxidation processes are useful methodologies to accomplish abatement of contaminants; however, elucidation of the reaction mechanisms is hampered by the difficult detection of the short-lived primary key species involved in the photocatalytic processes. Nevertheless, herein the combined use of an organic photocatalyst such as triphenylpyrylium (TPP + ) and photophysical techniques based on emission and absorption spectroscopy allowed monitoring the photocatalyst-derived short-lived intermediates. This methodology has been applied to the photocatalyzed degradation of different pollutants, such as acetaminophen, acetamiprid, caffeine and carbamazepine. First, photocatalytic degradation of a mixture of the pollutants showed that acetaminophen was the most easily photodegraded, followed by carbamazepine and caffeine, being the abatement of acetamiprid almost negligible. This process was accompanied by mineralization, as demonstrated by trapping of carbon dioxide using barium hydroxide. Then, emission spectroscopy measurements (steady-state and time-resolved fluorescence) allowed demonstrating quenching of the singlet excited state of TPP + . Laser flash photolysis experiments with absorption detection showed that oxidation of contaminants is accompanied by TPP + reduction, with formation of a pyranyl radical (TPP ), that constituted a fingerprint of the redox nature of the occurring process. The relative amounts of TPP detected was also correlated with the efficiency of the photodegradation process. [ABSTRACT FROM AUTHOR]

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI-LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI-LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI-LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI-LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls. [ABSTRACT FROM AUTHOR]

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability, and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine laser temperature-jump experiments and coarse-grained simulations to determine the folding/unfolding pathways of VPK, a variant of the mouse mammary tumor virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK serve as local probes allowing us to monitor the order of assembly of VPK that has two constituent hairpins with different intrinsic stabilities. We show that at 50 mM KCl, the dominant folding pathway populates only the more stable hairpin intermediate; as the salt concentration is increased, a parallel folding pathway emerges involving the less stable hairpin as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. Our findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. The experimental results of salt effects on the partitioning between the two folding pathways are in remarkable agreement with simulations that were performed with no adjustable parameters. Our study not only unambiguously demonstrates that VPK folds by parallel pathways but also showcases the power of combining experiments and simulations for a more enriched description of RNA self-assembly. [ABSTRACT FROM AUTHOR]