Fluorescence Emission Kinetics Research Articles

Caracterización del Cabernet-Sauvignon del Valle del Maipo (Chile) usando mediciones de fluorescencia

Virus infection in Vitis vinifera negatively impacts vineyard production and grape quality. Examples are grapevine leafroll-associated viruses (GLRaVs) and grapevine rupestris stem pitting-associated viruses (GRSPaV). We investigated transient fluorescence of chlorophyll a in leaves of Cabernet-Sauvignon, the primary red cultivar planted in Chile, using the OJIV test. First, we evaluated fluorescence in productive grapevines throughout their development, and then we analyzed the same parameters in plants infected with GLRaVs or GLRaVs+GRSPaV. We observed that throughout phenological development, as in the virus-affected clones, the kinetics of fluorescence emission decreased, particularly in values such as Fo and FM, reflecting the limitation in electron donation by the oxygen-releasing complexes and changes in the PSII antenna complex. The parameter reflecting the most significant differences was the performance index (PIABS), whose value decreases throughout phenological development and is also lower in virus-affected plants compared to healthy plants. This decrease would indicate that PSII changes the conversion of light energy of chemical energy to heat dissipation, making PIABS an excellent parameter to evaluate the vitality of Cabernet-Sauvignon vineyards.

Speckle patterning of a pumping laser light as a limiting factor for stimulated fluorescence emission in dense random media.

The features of fluorescence emission in a dye-doped dense multiple scattered medium under pulsed laser pumping are considered in terms of confined excitation in small zones associated with laser speckles occurring in a pumped medium. The results of numerical modeling of the fluorescence emission kinetics are compared to the experimental data obtained using the rhodamine 6G-doped layers of the densely packed TiO2 (anatase) particles pumped at 532 nm by 10 ns laser pulses. The intensity of pump radiation during the action of laser pulses was varied from 1·105 W/cm2 to 5·107 W/cm2. In the recovery of the ratios of stimulated to a spontaneous emission, the spectra of the stimulated component were fitted using the spectral function derived by R. Dicke. In the framework of the considered concept, saturation of the ratio of the stimulated to a spontaneous emission and linear growth of an integrated fluorescence output with a practically unchangeable half-width of the emission spectra at high pump intensities are interpreted.

Facile access to photo-switchable, dynamic-optical, multi-colored and solid-state materials from carbon dots and cellulose for photo-rewritable paper and advanced anti-counterfeiting

Dynamic optical materials can respond to external stimuli to give intriguing and chameleon-like visual effect and carry more information, they hence have attracted considerable attention. In this work, we propose a simple and efficient strategy to construct multi-color solid-state carbon dots (CDs) materials with photo-switchable and reversible dynamic-optical properties. A photochromic and positively-charged cellulose derivative C-Im+-SP with an imidazole salt (Im+) group and a spiropyran (SP) group is designed and synthesized. Subsequently, C-Im+-SP is spontaneously coated on the surface of negatively-charged CDs (NCDs) via electrostatic attraction to form a physical protective shell, which not only inhibits aggregation-caused quenching (ACQ) in NCDs but also promotes effective fluorescence resonance energy transfer (FRET). The resultant NCDs@C-Im+-SP exhibits a photo-triggering reversible dynamic-optical property. The NCDs to C-Im+-SP ratio can be used to effectively regulate fluorescence emission and fluorescence kinetics. The polymeric C-Im+-SP shell layer endows NCDs with excellent processability and formability, whereby NCDs@C-Im+-SP materials can be utilized as functional coatings and inks to fabricate photo-rewritable paper and dynamic anti-counterfeit patterns that have considerable potential applications for special coatings, complex anti-counterfeiting, and information encryption.

SENSITIVITY TO ENVIRONMENTAL STRESS OF PRATA,JAPIRA AND VITÓRIA BANANA CULTIVARS PROVEN BY CHLOROPHYLL a FLUORESCENCE

ABSTRACT This study aimed to evaluate the physiological responses to environmental stress during pre- and post-harvest of the following banana cultivars: Prata (AAB), Japira (AAAB) and Vitoria (AAAB). Analyses were carried out on young plants at vegetative stage (daughter-plant) and adult plants at reproductive stage (motherplant). The experimental design was completely randomized. In the in vivo pre-harvest analysis were used seven replications, in a factorial scheme (3x2x2), three cultivars and two stages (vegetative and reproductive) and two collection periods (March and June). For the analysis of post-harvest quality were used five replications in a factorial design (3x2x5), corresponding to three cultivars, two development stages and five periods of post-harvest analysis, carried out every two days from stage 4 of fruit ripening. The chlorophyll a fluorescence emission kinetics showed low photochemical performance of the three cultivars in June, a period characterized by lower temperatures and water deficit. Prata was the cultivar with the lowest tolerance to abiotic physiological behavior changes, which also reflected in fruit quality, because there was a change in physical and physicochemical parameters. Japira and Vitoria cultivars showed similar physiological responses in the pre- and post-harvest periods, according to their phylogenetic proximity. The total performance index, i.e., the conservation of energy absorbed by PSII up to the reduction of the final PSI acceptors (PItotal) and the di-malonic aldehyde (MDA) content were significantly higher in Japira and Vitoria cultivars compared to Prata cultivar in the reproductive phase. There was no significant change in the potential quantum efficiency of PSII (FV / FM = jP0) among the three cultivars. It was concluded that Japira and Vitoria cultivars showed greater plasticity to tolerate or even adapt to abiotic variations keeping higher fruit yield. PItotal is the most sensitive parameter during the banana life cycle and important tool for distinguishing different cultivars yields.

Dual Channel RESOLFT Nanoscopy by Using Fluorescent State Kinetics

We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues.

Rapid and subnanomolar assay of recombinant human erythropoietin by capillary electrophoresis using NanoOrange precolumn labeling and laser-induced fluorescence detection.

Because of less functionally critical carbohydrate sectors that contributed to the stability, efforts have been made to quantify intact recombinant human erythropoietin. A simple, rapid capillary electrophoresis with laser-induced fluorescence method for the assay of recombinant human erythropoietin was developed, with a limit of detection of intact recombinant human erythropoietin at subnanomolar concentration (up to 10 ng/mL or 3 × 10(-10) M), which is among the lowest reported. High sensitivity was accomplished by precolumn derivatization with the noncovalent dye NanoOrange. Capillary electrophoresis separation and reaction conditions were carefully manipulated for avoiding microheterogeneity of glycoforms and inhomogeneity of multiple labeling products. The fluorescence signal was linear over the range of 10 ng/mL-10 μg/mL, corresponding to the detection requirement of recombinant human erythropoietin in biofluids and pharmaceutical samples, as demonstrated by a real sample analysis. Although the salt in reaction mixtures showed a detrimental effect on the fluorescence of the derivatives, this method could tolerate a certain amount of salt, extending its application in biofluid analysis. In addition, zero-order fluorescence emission kinetics was obtained, indicating that the rapid decay of recombinant human erythropoietin was derived from a self-quenching effect.

Analyzing Structure–Photophysical Property Relationships for Isolated T8, T10, and T12 Stilbenevinylsilsesquioxanes

Silsesquioxanes (SQs) are of considerable interest for hybrid electronic and photonic materials. However, to date, their photophysical properties have not been studied extensively, thus their potential remains conjecture. Here we describe the first known efforts to map structure-photophysical properties as a function of cage symmetry and size by comparing identically functionalized systems. Our focus here is on the solution photophysical properties of the title stilbenevinyl-SQs, which were characterized using single photon absorption, two-photon absorption, fluorescence emission, and fluorescence lifetime kinetics. We offer here the first detailed photophysical study of the larger pure T10 and T12 silsesquioxanes and show photophysical properties that differ as a function of size, especially in their fluorescence behavior, indicating that cage size and/or symmetry can strongly affect photophysical properties. We also find that they offer excitation-dependent emission (evidence of rare "red-edge" effects). The T10 stilbenevinyl-SQ offers up to a 10-fold increase in two-photon absorption cross section per chromophore over a free chromophore, signifying increased electronic coupling. The SQ cage compounds show "rise times" of 700-1000 fs and low anisotropy (~0.1) in fluorescence lifetime kinetic studies. These results indicate excited state energy transfer, unobserved for the free chromophores and unexpected for systems with "inert" silica cores and for 3-D hybrid molecular species. These findings provide the first detailed photophysical study of chromophore-functionalized T10 and T12 silsesquioxanes and show that SQs may be considered a separate class of compounds/materials with anticipated novel properties of value in developing new components for electronic and photonic applications.

Fireflies‐On‐A‐Chip: (Ionic Liquid)–Aqueous Microdroplets for Biphasic Chemical Analysis

Inspired by the enchanting bioluminescence of fireflies, herein microfluidic droplet-based ‘fireflies’ for non-invasive biphasic chemical analysis are demonstrated. Bicompartmental droplets are created where a model analyte is transported from the aqueous compartment to the adjacent ionic liquid segment, yielding a bright fluid firefly structure via a localized catalytic reaction. Analysis of fluorescence emission kinetics and a method to passively decouple the two compartments at a constriction are presented.

Effect of metal cations on the fluorescence lifetimes of pyrene-labeled G-quadruplex probes

Abstract Fluorescence lifetime study of two probes abbreviated as Py-Htelom-Py and Py-TBA-Py, carrying pyrene moieties at both termini and sequences of human telomere and thrombin binding aptamer, respectively are reported. The effect of metal cations (sodium, potassium and strontium) on the photophysical processes was examined in order to elucidate factors that facilitate the production of excimer emission. The fluorescence spectra and emission kinetics data supported the conclusion that pyrene-quadruplex conjugate has multiple conformers in solution and that the relative orientation of pyrene and neighboring nucleobases (guanine, adenine, thymine) play a crucial role in determining both the rate of electron-transfer quenching of pyrene excited state and the efficiency of excimer emission.

In Vitro Fluorescence, Toxicity and Phototoxicity Induced by δ-Aminolevulinic Acid (ALA) or ALA-Esters

Synthesis of δ-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 μg/mL), E1 and E2 (10 μg/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 μg/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 μg/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Interesting fluorescence properties of C60-centered dendritic adduct with twelve symmetrically attached pyrenes.

A C60-centered dendritic adduct with 12 symmetrically attached pyrene species was synthesized and found to have relatively simple fluorescence emission kinetics, in particular, the mono-exponential decay of the significant pyrene excimer emission.

Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool: Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. These features, coupled with the requirement for high throughput assays in the clinical laboratory has prompted the development of two orthogonal NanoOrange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis laser-induced fluorescence (CE–LIF) assay formats. Dye performance in five commonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration generally decreases fluorescence emission and can be minimized by pre-diluting biological samples to a final salt concentration of 20–80 m M. Titration of protein with NanoOrange resulted in optimal HSA–NanoOrange complex formation utilizing 1× and 2× NanoOrange in the 96-well microplate and CE–LIF approaches, respectively. A NanoOrange binding model based on rapid signal enhancement and zero order fluorescence emission kinetics is proposed. The utilization of NanoOrange in CE–LIF based human serum analysis results in a signal-to-background ratio improvement of up to two orders of magnitude.

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a ≈ 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (τ) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (τ3 and τ4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of τ3 and in the average lifetime (≈ 2.4 ns with zeaxanthin and 3.2–3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2–5.3) is observed on chlorophyll a fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters.

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Thermostable lipase of Bacillus Stearothermophilus: high-level production, purification, and calcium-dependent thermostability.

An efficient expression system was developed for the production of the thermostable lipase from Bacillus stearothermophilus L1 in an Escherichia coli system. A structural gene corresponding to mature lipase was subcloned in the pET-22b(+) expression vector and its expression was induced by IPTG at 30 degrees C in E. coli cells. The lipase activity in a cell-free extract was as high as 448,000 units/g protein, which corresponds to as much as 26% of the total cellular protein and is 77 times higher than that of E. coli RR1/pLIP1. Based on its pI (7.4) and pH stability data reported previously, the L1 lipase was efficiently purified to homogeneity with CM (at pH 6.0) and DEAE (at pH 8.8) column chromatographies with a recovery yield of 62%. The specific activity of the purified enzyme was 1700 units/mg protein when olive oil emulsion was used as a substrate. Its optimum temperature for the hydrolysis of olive oil was 68 degrees C and it was stable up to 55 degrees C for 30 min-incubation. The thermostability increased by about 8-10 degrees in the presence of calcium ions. This calcium-dependent thermostability was confirmed by the tryptophan fluorescence emission kinetics showing that the enzyme starts to unfold at 66 degrees C in the presence of calcium ions but at 58 degrees C in the absence of calcium ions, implying that the calcium ions bind to the thermostable enzyme and stabilize the protein tertiary structure even at such high temperatures.

Biomolecular electron transfer under high hydrostatic pressure

The dependence of the photoinduced electron transfer rate on hydrostatic pressure up to 8 kbar was studied at 295 K in a bridged Zn-porphyrin donor and pyromellitimide acceptor supermolecule dissolved in toluene. A picosecond fluorescence emission kinetics of the donor, limited by the electron transfer rate, was detected by using synchroscan streak camera. The experiment was complemented with model calculations based on modified classical and semi-classical nonadiabatic electron transfer theory. A peculiar asymmetric inverted parabola-like dependence of the electron transfer rate on pressure was observed. The dependence was successfully reproduced by nonadiabatic theory in the high-temperature limit assuming that the reorganisation free energy or both the reorganisation free energy and the reaction driving force (linearly) changed with pressure. The reaction driving force dependence on pressure alone failed to explain the asymmetry, suggesting that the electron transfer was accompanied with vibration frequency changes. It was inferred that the effective frequency in the product state should be larger than in the reactant state. The usage of the nonadiabatic theory is well justified due to the fulfilment of the inequalities V《 k B T and V《〈 τ L〉 −1 ( V is the electronic coupling matrix element, 〈 τ L〉 −1 is the solvent relaxation rate). The influence of the donor-acceptor distance reduction under compression on the electron transfer rate was found to be minor.

Fluorescence heterogeneity of tryptophans in Na,K-ATPase: evidences for temperature-dependent energy transfer

The intrinsic fluorescence emission kinetics of Na,K-ATPase, a large membrane protein containing 16 tryptophan residues, was studied by time-resolved techniques. The lifetime distributions recovered by the Maximum Entropy Method exhibit a strong dependence on the emission wavelength at temperatures between 37°C and −70°C. From the `blue' edge of the fluorescence emission spectrum up to the maximum of emission, the lifetime distribution at room temperature is the result of four broad peaks which cover the time range 0.3–7 ns. With increasing emission wavelength, these peaks move to longer lifetimes and the peak at shorter times are suppressed at the red edge, while the longest component (6–7 ns) becomes dominant. With decreasing temperature, the number of lifetime components is reduced for the benefit of the long one. At cryogenic temperatures, the emission decay in the red-edge of the fluorescence spectrum consists of one major slow component (6–7 ns) and a fast one (0.5 ns) associated with a negative pre-exponential term. This is a characteristic feature of an excited-state reaction. The temperature dependence of this fast component and the fluorescence anisotropy decay at low temperature in the red-edge, indicate that this excited state reaction may be accounted for a unidirectional inter-tryptophan fluorescence energy transfer from `blue' populations of donors to `red' populations of acceptors. This is also illustrated by the time-resolved emission spectra. In the blue edge of the fluorescence emission spectrum, moreover, the time course of the anisotropy decay suggests the existence of homo-transfer of excitation energy involving `blue' tryptophan residues. The steady-state anisotropy excitation spectrum in vitrified solvent agrees with this suggestion. These different energy transfer mechanisms may be used as structural probes to detect more accurately conformational changes of the protein elicited by effectors and ion binding or release.

SOMATOSTATIN IN A WATER‐RESTRICTED ENVIRONMENT: FLUORESCENCE AND CIRCULAR DICHROISM STUDY IN AOT REVERSE MICELLES

Steady-state and time-resolved fluorescence emission from the single tryptophan residue of somatostatin, and the kinetics of quenching of this emission, were studied in aqueous solution and in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT)/water/isooctane, a system that mimics the water-membrane interface well. Incorporation into micelles caused blue shifts and reduced band-widths of the emission peaks and altered the quantum yields with respect to emission from bulk water. Steady-state anisotropy values also increased considerably on micellization. These observations point to reduced polarity of the environment around the Trp residue of the peptide, as well as restricted freedom of its rotational motions, due to transfer from the aqueous to the micellar phase. Fluorescence emission kinetics of the Trp moiety followed biexponential decay laws in both aqueous and micellar media. Static and dynamic quenching constants were measured for acrylamide and CCI4 quenchers localized in the micellar and organic pseudophases, respectively, using both steady-state and time-resolved experiments. The efficiency of dynamic quenching by acrylamide became vanishingly small in going from water to reverse micelles, in sharp contrast to the comparable quenching efficiencies exhibited by CCI4 in micelles and acrylamide in water. The circular dichroic (CD) spectrum of the native peptide in water indicated the possibility of some amount of beta-type secondary structure being present. Conformational analysis of CD spectra in micelles showed that the relative amount of this structural feature was enhanced for the micellized peptide but was insensitive to the water content of micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the cation-induced reversible changes in excitation energy distribution in thylakoids of BASF 13.338-grown plants

Measurements of relative quantum yields of Photosystem II and Photosystem I partial reactions and room-temperature chlorophyll a fluorescence of thylakoids in high- and low-salt media showed that the cation-induced changes in the excitation energy distribution were inhibited in the thylakoids isolated from pea plants grown in the presence of sublethal concentration of the pyridazinone herbicide BASF 13.338. Simultaneous measurement of Photosystem II and Photosystem I fluorescence emission kinetics at 77 K showed that the ability of cations to regulate excitation energy spillover from Photosystem II to Photosystem I was inhibited in thylakoids of the BASF-grown plants. Cation regulation of the absorption cross-section of the photosystems was not affected. Electron microscope data revealed that the proportion of stroma membranes relative to grana membranes was markedly less in the thylakoids of the BASF-grown plants. Furthermore, when the thylakoids were resuspended in low-salt medium, no unstacking of the grana was detected. The observed inhibition of cation-induced spillover change was presumably due to loss of ability of these photosynthetic membranes to undergo unstacking in low-salt medium.