Fluorescence Emission Measurements Research Articles

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity.

This study provides a full description of the responses of the crop energy plant Zea mays to stress induced by Cd and Pb, in view of a possible extensive use in phytoattenuation of metal-polluted soils. In this perspective, (i) the uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency based on photochemistry and the level of key proteins were investigated in hydroponic cultures. Both metals were uptaken by maize, with a translocation factor higher for Cd than Pb, but only Cd-treated plants showed a reduced growth compared to control (i.e., a lower leaf number and a reduced plant height), with a biomass loss up to 40%, at the highest concentration of metal (10-3M). The observation of cytological traits highlighted ultrastructural damages in the chloroplasts of Cd-treated plants. A decline of Rubisco and D1 was observed in plants under Cd stress, while a relevant increase of the same proteins was found in Pb-treated plants, along with an increase of chlorophyll content. Fluorescent emission measurements indicated that both metals induced an increase of NPQ, but only Cd at the highest concentration determined a significant decline of Fv/Fm. These results indicate a different response of Z. mays to individual metals, with Pb triggering a compensative response and Cd inducing severe morpho-physiological alterations at all investigated levels. Therefore, Z. mays could be successfully exploited in phytoattenuation of Pb-polluted soil, but only at very low concentrations of Cd to avoid severe plant damages and biomass loss.

Effect of Ionic Liquids as Cosurfactants on Photoinduced Electron Transfer in Tetronic Micelles.

This study investigates the role of varying alkyl chain lengths of a series of surface-active 1-alkyl-3-methylimidazolium tetrafluoroborate ([C nMIm][BF4], n = 4, 6, and 10) ionic liquids (ILs) as cosurfactants in modifying the micellar characteristics of a tetronic star-block copolymer, T1304, and the consequent effects on bimolecular photoinduced electron transfer (PET) reactions carried out in these T1304-IL mixed micellar systems. Using coumarin 153 as the probe dye and following ground-state absorption, steady-state fluorescence, and time-resolved emission measurements, the micropolarity, microviscosity, and solvent relaxation dynamics in the micellar palisade layer have been revealed both in pure T1304 and in T1304-IL systems. With increasing alkyl chain length of the ILs, the palisade layer of the micelles gradually becomes more polar and less viscous, suggesting better incorporation of the longer alkyl chain length ILs as cosurfactants into the T1304 micelles. The bimolecular PET reactions, involving 7-aminocoumarins as acceptors and N, N-dimethylaniline as the donor, are considerably modulated in T1304 micelles by the presence of the ILs, the effect being more prominent for ILs with longer alkyl chain lengths. In all of the micellar systems, correlations of the electron transfer (ET) kinetics with the reaction exergonicity (-Δ G0) show clear Marcus inversion (MI) behavior where onsets of MI invariably appear at significantly lower exergonicities, suggesting the involvement of a two-dimensional ET mechanism. Interestingly, the Marcus correlations display significant variations, namely, enhanced reaction rates and gradual shift in the onset of MI toward higher exergonicity, as longer alkyl chain length ILs are sequentially introduced as cosurfactants. From the observed results, it is convincingly realized that 1-alkyl-3-methylimidazolium-based ILs can be used satisfactorily as cosurfactants in tetronic star-block copolymer solutions to modulate PET reactions very significantly for their better utilizations in suitable applied areas.

An excitation emission fluorescence lifetime spectrometer using a frequency doubled supercontinuum laser source

The accurate fluorescence analysis of complex, multi-fluorophore containing proteins requires the use of multi-dimensional measurement techniques. For the measurement of intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) one needs tuneable UV excitation and for steady-state measurements like Excitation Emission Matrix (EEM) simple pulsed Xe lamps are commonly used. Unfortunately, simultaneous multi-dimensional wavelength and time resolved measurement of intrinsic protein fluorescence in the 260 to 400 nm spectral range are challenging and typically required the use of very complex tuneable laser systems or multiple single excitation wavelength sources. Here we have assembled and validated a novel Excitation Emission Fluorescence Lifetime Spectrometer (EEFLS) using a pulsed, frequency doubled, Super-Continuum Laser (SCL) source coupled with a 16 channel multi-anode Time Correlated Single Photon Counting (TCSPC) measurement system. This EEFLS enabled the collection of near complete lifetime and intensity maps over the most important intrinsic protein fluorescence spectral range (λex = 260–350/λem = 300–500 nm). The 4-dimensional (λex/λem/I(t)/τ) Excitation Emission Fluorescence Lifetime Matrix (EEFLM) data produced can be used to better characterize the complex intrinsic emission from proteins. The system was capable of measuring fluorescence emission data with high spectral (1–2 nm) resolution and had an Instrument Response Function (IRF) of ∼650 ps for accurate measurement of nanosecond lifetimes. UV power output was stable after a warm up period, with variations of <2% over 9 hours and reproducible (relative standard deviation RSD < 1.5%). This enabled the collection of accurate EEFLM data at low resolution (∼12 nm in excitation and emission) in 1–2 hours or high resolution (4 nm) in ∼17 hours. EEFLS performance in the UV was compared with a conventional commercial TCSPC system using pulsed LED excitation and validated using solutions of p-terphenyl and tryptophan.

Detail Photophysical Studies of Sulfonated Polyaniline in Aqueous Medium.

Sulfonated polyaniline (SPANI) has emerged as a promising polymer in the past few decades due to its solubility in water and relatively moderate conductivity. However, to date, literature data on the optical characterization of SPANI are very limited and preliminary in nature. In the present work, SPANI is synthesized by direct sulfonation of emeraldine salt form of polyaniline with chlorosulfonic acid in an inert solvent. Detail photophysical properties of SPANI are investigated in aqueous medium by using steady state (concentration, temperature, pH, and excitation wavelength dependence) and time-resolved spectroscopic techniques. The steady state fluorescence emission measurements are carried out carefully to avoid inner filter effect (especially secondary inner filter effect or reabsorption effect) as well as scattering. Two ground state conformations of SPANI are suggested to exist in aqueous medium. Excitation wavelength dependence of the fluorescence emission spectra is attributed to red-edge effect. All these observations are nicely corroborated by the fluorescence lifetime data of SPANI obtained from time-resolved measurements. All these new findings are extremely important in view of the potential applications of SPANI in polymer optoelectronics.

Versatile Approach for Reliable Determination of Both High and Low Values of Luminescence Quantum Yields.

The determination of luminescence quantum yields by means of relative methods requires setting identical experimental conditions for both sample and reference compounds. This requirement has a critical impact on the applicability of these protocols, as it does not allow for the precise determination of low quantum-yield values using well-characterized high-quantum-yield standards. We show that using the simultaneous absorption and fluorescence-emission measurement (SAFE) approach [ Nawara and Waluk. Anal. Chem. 2017 , 89 , 8650 ], the sample and reference compounds can be effectively measured with different excitation-slit spectral bandpass or integration times, separately optimized for each chromophore. This unique feature simplifies the determination of luminescence quantum yields, allowing measurements of low quantum-yield values using well-characterized high-quantum-yield standards.

Nonradiative energy transfer and ion-ion interactions in terbium-praseodymium coactivated borate glass

A study of energy transfer between Tb3+ and Pr3+ in borate glass is being presented. From our measurement of fluorescence emission and decay times of donor (Tb3+), in the presence of different concentrations of acceptor (Pr3+), the energy transfer probability (Pda) and transfer efficiency (ɳT) have been calculated. Analyzing the data it is observed that the non-radiative energy transfer in our borate glass system occurs by dipole-dipole interactions, contrary to the observations made by Nakazawa and Shionoya (dipole-quadrupole interaction) in phosphate glass. A linear dependence of Pda versus C at low acceptor concentration suggests the migration of excitation energy among donor ions before the energy is finally transferred to the acceptor ion. At high acceptor concentrations a plot of Pdavs (C0 + C)2; where C0 is donor concentration and C is the acceptor concentration, presents a linear dependence suggesting thereby that one donor interacts with two neighbouring acceptors consistent with Fong-Diestler mechanisms of dipole-dipole energy transfer interactions.

An Air-Stable Organoboron Compound, Dithienooxadiborepine: Preparation and Functionalization.

π-Conjugated organoboron molecules, which are easy to prepare, stable against moisture, and easy to functionalize, are scarce. Here, we report a one-pot synthesis of an air-stable organoboron compound, dithienooxadiborepine 1 in 17% yield on a 600 mg scale without separation or handling an air-sensitive intermediate. Dithienooxadiborepine 1 showed excellent stability under ambient conditions, allowing conventional column chromatography purification. Functionalization of 1 was realized via direct bromination using NBS and further Stille coupling reactions, giving access to longer π-conjugated molecules 5A and 5B. Single-crystal structures of compounds 4, 5A, and 5B not only unambiguously verified the chemical identity of dithienooxadiborepine 1 but also revealed that both the seven-member oxadiborepine ring and the 5-7-5 fused dithienooxadiborepine ring system are planar. UV-vis absorption and fluorescence emission measurements of 5A and 5B showed bathochromic shifted absorption and emission relative to 1, evidencing good π-conjugation. Cyclic voltammograms of 5A and 5B displayed two reduction peaks corresponding to two electron-accepting events at two boron atoms. These results proved dithienooxadiborepine 1 a potent π-conjugating building block for electron-accepting materials.

Selective detection of Co2+ by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and biological activities

Nowadays scientist over the world are engaging to put forth improved methods to detect metal ion in an aqueous medium based on fluorescence studies. A simple, selective and sensitive method was proposed for detection of Co2+ ion using fluorescent organic nanoparticles. We synthesized a fluorescent small molecule viz. 4,4′-{benzene-1,4-diylbis-[(Z)methylylidenenitrilo]}dibenzoic acid (BMBA) to explore its suitability as sensor for Co2+ ion and biocompatibility in form of nanoparticles. Fluorescence nanoparticles (BMBANPs) prepared by simple reprecipitation method. Aggregation induced enhanced emission of BMBANPs exhibits the narrower particle size of 68nm and sphere shape morphology. The selective fluorescence quenching was observed by addition of Co2+ and does not affected by presence of other coexisting ion solutions. The photo-physical properties, viz. UV-absorption, fluorescence emission, and lifetime measurements are in support of ligand-metal interaction followed by static fluorescence quenching phenomenon in emission of BMBANPs. Finally, we develop a simple analytical method for selective and sensitive determination of Co2+ ion in environmental samples. The cell culture E. coli, Bacillus sps., and M. tuberculosis H37RV strain in the vicinity of BMBANPs indicates virtuous anti-bacterial and anti-tuberculosis activity which is of additional novel application shown by prepared nanoparticles.

Determination of PS I oligomerisation in various cyanobacterial strains and mutants by non-invasive methods

PSI trimer to monomer ratio in intact cyanobacterial cells and isolated thylakoids was analysed by two noninvasive, in vivo methods; low-temperature fluorescence emission and circular dichroism spectroscopy. We measured fluorescence emission spectra of cells upon chlorophyll (Chl, 436 nm) excitation. All three species – Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Spirulina platensis – showed shifted Chl peak, indicating they have different spectral properties. CD spectroscopy revealed the highest intensity at 515 nm (PSI peak) in Spirulina platensis cells, which may originate from PSI multi-oligomerisation. The most sensitive response to heat treatment in this strain was the oligomerisation of PSI RCs. PSI dimers and tetramers in Anabaena cells showed smaller changes of the CD signal upon the heat treatment compared to that of Synechocystis WT. The lack of γ-linolenic acid affected the filament morphology by the loss of the spiral shape and the PSI monomerisation in Spirulina I22.

Construction of uranyl phosphonates from multifunctional zwitterionic ligands

Abstract Two new uranyl diphosphonates, namely, [UO2(H3L1)2(H2O)]·4H2O (1) and [UO2(H3L2)(H2O)]·3H2O (2), H4L1 = PyNHCH(PO3H2)2, H5L2 = Py(COOH)NHCH(PO3H2)2, have been hydrothermally synthesized from the reactions of two diphosphonate ligands with uranyl nitrate hexahydrate and characterized systematically using powder and single-crystal X-ray diffraction, IR, EA, TGA and fluorescent emission measurements. The proton transfer from the phosphonic acid groups to the pyridine and imine groups leads to the formation of zwitterionic ligands. The diphosphonate ligand in compound 1 adopts chelate coordination mode resulting in molecular structure while displays chelate-bridging coordination mode in compound 2 leading to the formation of 1D infinite chain structure. The fluorescent properties of compounds 1 and 2 were also investigated.

Improved Method of Fluorescence Quantum Yield Determination.

In the most widely used procedure for luminescence quantum yield determination, absorption and emission spectra are measured on two different instruments. This leads to errors caused by wavelength misalignment and different monochromator characteristics of the spectrophotometer and the spectrofluorometer. These errors can be avoided using a method for fluorescence quantum yield determination that relies on simultaneous absorption and fluorescence emission (SAFE) measurement using a single commercial spectrofluorometer. The method's performance is compared with the standard routinely used procedure for the relative quantum yield determination. The advantages of SAFE measurement are discussed. The proposed novel approach eliminates a number of potential errors in quantum yield determination protocol and provides higher accuracy.

Changes in aggregation states of light-harvesting complexes as a mechanism for modulating energy transfer in desert crust cyanobacteria

In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.

Design and synthesis of the BODIPY-BSA complex for biological applications.

A quinoxaline-functionalized styryl-BODIPY derivative (S1) was synthesized by microwave-assisted Knoevenagel condensation. It exhibited fluorescence enhancement upon micro-encapsulation into the hydrophobic cavity of bovine serum albumin (BSA). The S1-BSA complex was characterized systematically using ultraviolet (UV)-visible absorption, fluorescence emission, kinetics, circular dichroism and time-resolved lifetime measurements. The binding nature of BSA towards S1 was strong, and was found to be stable over a period of days. The studies showed that the S1-BSA complex could be used as a new biomaterial for fluorescence-based high-throughput assay for kinase enzymes.

Quantification of the Depolarization and Anisotropy of Fluorophore Stokes-Shifted Fluorescence, On-Resonance Fluorescence, and Rayleigh Scattering.

Fluorophores are important but optically complicated photonic materials as they are simultaneous photon absorbers, emitters, and scatterers. Existing studies on fluorophore optical properties have been focused almost exclusively on its photon absorption and Stokes-shifted fluorescence (SSF) with scant information on the fluorophore photon scattering and on-resonance fluorescence (ORF). Presented herein is a unified theoretical framework and experimental approach for quantification of the fluorophore SSF, ORF, and scattering depolarization and anisotropy using a combination of fluorophore UV-vis, fluorescence emission, and resonance synchronous spectroscopic spectral measurements. A mathematical model for calculating fluorophore ORF and scattering cross sections has been developed that uses polystyrene nanoparticles as the external reference. The fluorophore scattering cross section is ∼10-fold smaller than its ORF counterparts for all the six model fluorophores, but more than 6 orders of magnitude larger than the water scattering cross section. Another finding is that the fluorophore ORF has a depolarization close to 1, while its Rayleigh scattering has zero depolarization. This enables the experimental separation of the fluorophore ORF and photon scattering features in the fluorophore resonance synchronous spectra. In addition to opening a new avenue for material characterization, the methods and insights derived from this study should be important for developing new analytical methods that exploit the fluorophore ORF and photon scattering properties.

Aldoxime based biphenyl-azo derivative for self-assembly, chemosensor (Hg2+/F−) and bioimaging studies

A versatile bifunctional biphenyl-azo conjugated based aldoxime (DOXPA) was developed and its photo-responsive behaviours upon the addition of analytes has been investigated using UV–vis absorption, fluorescence emission, quantum yield and fluorescence lifetime measurements. The probe selectively recognized Hg2+ and F− over a wide range of metal ions/anions through “turn-on” fluorogenic modes. The nature of interaction between probe and Hg2+/F− has been studied using NMR titrations and DFT studies. Further, the probe has been successfully applied for intracellular sensing and imaging of Hg2+ and F−. Furthermore, we obtained sheet like morphology upon self-assembly of azo-derivative, due to the formation of hydrogen bonding between hydroxylamine. This was confirmed by Scanning Electron Microscope and Atomic Force Microscopy.

Electronic setup for fluorescence emission measurements and long-time constant-temperature maintenance of Single-Walled Carbon Nano-Tubes in water solutions

Abstract In our previous research we have observed that the fluorescence emission from water solutions of Single-Walled Carbon Nano-Tubes (SWCNT), excited by a laser with a wavelength of 830nm, diminishes with the time. We have already proved that such a fading is a function of the storage time and the storage temperature. In order to study the emission of the SWCNT as a function of these two parameters we have designed and realized a special measurement compartment with a cuvette holder where the SWCNT solutions can be measured and stored at a fixed constant temperature for periods of time as long as several weeks. To maintain the measurement setup under a constant temperature we have designed special experimental setup based on two Peltier cells with electronic temperature control.

Light/temperature dual-responsive ABC miktoarm star terpolymer micelles for controlled release

A novel light- and temperature-responsive amphiphilic ABC miktoarm star terpolymer consisting of hydrophilic poly(ethylene glycol) (PEG), light-responsive poly(2-nitrobenzyl methacrylate) (PNBM), and temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) arms, PEG(-b-PNBM)-b-PNIPAM, was synthesized via a combination of atom transfer radical polymerization (ATRP) and click reaction. In aqueous solution, the obtained miktoarm star terpolymer self-assembled into spherical micelles with PNBM cores and hybrid PEG/PNIPAM coronas. When heating above the phase transition temperature of PNIPAM, larger aggregates with mixed PNBM/PNIPAM cores and PEG coronas were formed. Upon UV light irradiation, the initial micelles were disrupted and reassembled into some large micelle complexes. Fluorescence emission measurements showed that Nile red encapsulated in the polymeric micelles could be released by stimuli of UV light or temperature change. More interestingly, the UV triggered release could be strengthened in basic conditions but weakened in acidic conditions. The prepared light- and temperature-responsive polymeric micelles may have great potential in applications such as nanocarriers and biotechnology for controlled drug and bioagent delivery.

Characterization of WY 14,643 and its Complex with Aldose Reductase.

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.

Impact of the aggregation behaviour of sodium cholate and sodium deoxycholate on aqueous solution structure and dynamics: A combined time resolved fluorescence and dielectric relaxation spectroscopic study

The impact of aggregation behaviour of sodium cholate (SC) and sodium deoxycholate (SDC) on aqueous solution structure and dynamics has been studied and compared by performing dielectric relaxation (DR) and time-resolved fluorescence (TRF) measurements at three different concentrations, 30mM, 100mM and 300mM at ~298K. For DR studies, measurements have been carried out in the frequency range, 0.2≤ν(GHz)≤50. TRF measurements, on the other hand, employ a fluorescent probe, coumarin 153 (C153), as a local reporter. Both DR and TRF measurements confirm presence of surface water molecules that are characterized by sub-nanosecond timescale. This is further supported by the sub-melting features in the differential scanning calorimetric measurements. DR, dynamic Stokes shift and fluorescence anisotropy experiments indicate SDC solutions are dynamically slower than SC solutions. In addition, excitation energy dependence of steady state fluorescence emission measurements suggests SDC solutions are more heterogeneous. All these results indicate that absence of only one hydroxyl (−OH) group in SDC introduces significant difference in the aggregation behaviour between SC and SDC, affecting substantially the solution structure and dynamics.

Coexistence of Different Electron-Transfer Mechanisms in the DNA Repair Process by Photolyase.

DNA photolyase has been the topic of extensive studies due to its important role of repairing photodamaged DNA, and its unique feature of using light as an energy source. A crucial step in the repair by DNA photolyase is the forward electron transfer from its cofactor (FADH(-) ) to the damaged DNA, and the detailed mechanism of this process has been controversial. In the present study, we examine the forward electron transfer in DNA photolyase by carrying out high-level ab initio calculations in combination with a quantum mechanical/molecular mechanical (QM/MM) approach, and by measuring fluorescence emission spectra at low temperature. On the basis of these computational and experimental results, we demonstrate that multiple decay pathways exist in DNA photolyase depending on the wavelength at excitation and the subsequent transition. This implies that the forward electron transfer in DNA photolyase occurs not only by superexchange mechanism but also by sequential electron transfer.