Home About us Contact
|
Home About us Contact | ||
|
|
||
Fluorescence Decay (fluorescence + decay)
Selected AbstractsMonitoring Protein Interactions in the Living Cell Through the Fluorescence Decays of the Cyan Fluorescent ProteinCHEMPHYSCHEM, Issue 7 2006Regis Grailhe Dr. Abstract Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33,% average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP,YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies. [source] Severing of F-actin by yeast cofilin is pH-independentCYTOSKELETON, Issue 9 2006Dmitry Pavlov Abstract Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, ,-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by ,ATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2,3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green® 488 maleimide. The depolymerization of actin by cofilin was faster at high pH. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Synthesis and Optoelectronic Properties of Nonpolar Polyrotaxane Insulated Molecular Wires with High Solubility in Organic Solvents,ADVANCED FUNCTIONAL MATERIALS, Issue 21 2008Michael J. Frampton Abstract Hydrophilic polyanionic conjugated polyrotaxanes are readily synthesized in water by Suzuki coupling, but their high polarity and ionic nature limit the potential applications of these materials. Here, we demonstrate three methods for transforming these polar polyelectrolytes into nonpolar lipophilic insulated molecular wires. A water-soluble polyfluorene- alt -biphenylene ,-cyclodextrin (CD) polyrotaxane was converted into nonpolar derivatives by methylation of the carboxylic acid groups with diazomethane and conversion of the hydroxyl groups of the CDs to benzyl ethers, trihexylsilyl ethers, benzoyl esters, and butanoate esters to yield polyrotaxanes that are soluble in organic solvents such as chloroform and cyclohexane. Elemental analysis, NMR spectroscopy, and gel permeation chromatography (GPC) data support the proposed structures of the organic-soluble polyrotaxanes. The extents of reaction of the polyrotaxane CD hydroxyl groups were 55% for trihexylsilyl chloride/imidazole; 81% for benzyl chloride/sodium hydride; 72% for benzoyl chloride/pyridine/4-dimethylaminopyridine; and 98% butanoic anhydride/pyridine/4-dimethylaminopyridine. Alkylation, silylation, and esterification increase the bulk of the encapsulating sheath, preventing interstrand aggregation, increasing the photoluminescence efficiency in the solid state and simplifying the time-resolved fluorescence decay. The organic-soluble polyrotaxanes were processed into polymer light-emitting diodes (PLEDs) from solution in nonpolar organic solvents, thereby excluding ionic impurities from the active layer. [source] Scavenging of reactive oxygen species by the plant phenols genistein and oleuropeinLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2005Irena Kruk Abstract The plant-derived phenolic compounds genistein and oleuropein are known to exhibit several biological properties, many of which may result from their antioxidant and free radical scavenger activity. In this paper we report the results of a complex study of antioxidant activity of genistein and oleuropein, using electron spin resonance (ESR), chemiluminescence, fluorescence and spectrophotometric techniques. Different reaction systems were applied to study the inhibitory effect of the phenolic compounds studied: (a) the potassium superoxide[sol ]18- crown -6 dissolved in DMSO system, which generates superoxide radical (O2·,) and hydrogen peroxide (H2O2); (b) the Co(II),EDTA,H2O2 system (the Fenton-like reaction), which generates hydroxyl radical (HO·); (c) 2,2,-azobis(2-amidino-propane)dichloride (AAPH) as the peroxyl radical (ROO·) generator, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. Results showed that genistein and oleuropein decreased the chemiluminescence sum from the O2·, generating system, an inhibitory effect that was dependent on their concentration. These compounds also reacted with ROO radicals and they showed activity about two-fold greater than the standard Trolox. The antioxidant effects were studied at different concentrations and reflected in protection against the fluorescence decay of , -phycoerythrin (, -PE), due to ROO· attack on this protein. Using the Fenton-like reaction and the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the phenolic compounds examined were found to inhibit DMPO,·OH radical formation in the range 10,90% at concentrations of 0.1 mmol[sol ]L to 2 mmol[sol ]L. Furthermore, these compounds also inhibited HO· -dependent deoxyribose degradation; about 20% and 60% inhibitions were observed in the presence of 0.5 mmol[sol ]L genistein and oleuropein, respectively. It was also demonstrated that genistein had a weaker DPPH radical scavenging activity than oleuropein. Our results confirm good scavenging activity towards O2·,, HO· and ROO· and the antioxidant effect of genistein and oleuropein. Copyright © 2005 John Wiley & Sons, Ltd. [source] The effect of fungal metabolites on leaves as detected by chlorophyll fluorescenceNEW PHYTOLOGIST, Issue 2 2001Anandini Kshirsagar Summary ,,The effect is reported here of cytochalasin E isolated from the fungus Rosellinia necatrix on photosynthesis in young leaves of Malus domestica (apple). ,,Cytochalasin E was administered via the petiole to excised leaves. The chlorophyll fluorescence emission spectrum and time resolved fluorescence decay were measured up to the point where visible leaf damage was observed. ,,Within 2 h, the ratio of fluorescence emission at 730 nm decreased with respect to the peak at 690 nm. Over 6 h a small blue shift in the 690 nm emission band to 685 nm was seen. The time resolved fluorescence decay showed changes over a similar timescale after administration of cytochalasin E. The control decay could be fitted by two components, ,1, 112 ps, ,2, 402 ps, but after 6 h treatment with cytochalasin E the decay required a further component ,3, 4.25 ns for a good fit. ,,Cytochalasin E has a direct effect on photosynthesis, possibly as a result of impairment of light harvesting. This might partially account for the pathogenicity of the root infecting R. necatrix. Fluorescence techniques may therefore provide an early detection system for the fungus, a necessary prerequisite for development of a control strategy. [source] Fusion,Fission Transport of Probes and Quenchers in Microdomains of an Amphiphilic Ionene Polyelectrolyte,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2007Celize M. Tcacenco In aqueous solution, amphiphilic ionenes such as the [3,22]-ionene spontaneously adopt globular conformations and form microdomains that are highly micelle-like, i.e. are capable of solubilizing organic molecules, binding and exchanging counterions and accelerating or inhibiting the rates of bimolecular reactions. Time-resolved fluorescence decay of pyrene and pyrene derivatives solubilized in these microdomains at concentrations where excimer formation occurs show that even water-insoluble probes can migrate between the hydrophobic microdomains formed in aqueous solution by a [3,22]-ionene chloride (with the N-terminal groups quaternized with benzyl chloride). Time-resolved studies of the quenching of pyrene fluorescence by alkylpyridine derivatives revealed similar behavior. The observed quenching behavior requires that the migration be between microdomains on the same ionene chain or same group of associated ionene chains and is consistent with migration dominated by fusion/fission transport of the probe and quencher. [source] Time-resolved Microspectrofluorimetry and Fluorescence Lifetime Imaging of Hypericin in Human Retinal Pigment Epithelial Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2005Paola Taroni ABSTRACT Hypericin is the active ingredient of the off-the-shelf antidepressant St. John's Wort. It is an effective phototoxic agent and its systemic administration at therapeutic doses could induce particular damage in the eye due to continuous light exposure. Hypercin is strongly fluorescent and its fluorescence properties can be monitored to investigate noninvasively its localization and interactions. To this aim, time-resolved microspectrofluorimetry and fluorescence life-time imaging were used to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by retinal pigment epithelium (RPE) cells treated with Hyp at concentrations in the micromolar range (0.5,10 ,M). In the presence of hypericin, the emission peaks at 600-605 nm and the fluorescence decay is best fitted with three lifetimes (5.5-7 ns, 1.9-2.5 ns and < 0.8 ns). Spectral and temporal differences were observed between high (,5 ,M) and low hypericin concentrations. In particular, upon increasing concentration, the emission spectrum of the slow component broadens and its lifetime shortens. The latter change is observed also when high concentrations are reached locally, due to more efficient localization within the cell. [source] Two mechanisms of 1D2 fluorescence quenching of Pr3+ -doped Y2SiO5 crystalPHYSICA STATUS SOLIDI (B) BASIC SOLID STATE PHYSICS, Issue 3 2003Yu. V. Malyukin Abstract In Y2SiO5:Pr3+ crystals (0.1 at%, 0.3 at%, 0.6 at% , and 1.8 at% Pr3+) characterized by the existence of two types of Pr3+ optical centers, the energy transfer has been investigated using time-resolved site-selective spectroscopy techniques. The results obtained show that at certain conditions there are two different mechanisms of fluorescence quenching of the excited 1D2 states of Pr3+ ions. At 0.3 at% Pr3+ and under the excitation of 1D2 states as the result of 3P0 , 1D2 nonradiative relaxation, the phonon-assisted energy transfer from 1D2 states of the I-type Pr3+ ions to the II-type ones has been found. The nonexponential part of donor fluorescence decay was described by the law t0.5. and the transfer efficiency exhibits a strong temperature dependence in the range of 1.5,80 K. At the direct selective excitation of the 1D2 states of one type Pr3+ optical centers it was possible to observe only their own fluorescence which quenched at a concentration above 0.6 at% Pr3+. The donor fluorescence decay was not fit by the law t0.5 and the quenching efficiency was characterized by the square-loaw dependence on the concentration and a very poor dependency on the temperature. The analysis of some models allows us to assume, that in this case, the cooperative quenching of the 1D2 states of both type Pr3+ optical centers can take place. (© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] New insight on ,-lactoglobulin binding sites by 1-anilinonaphthalene-8-sulfonate fluorescence decayPROTEIN SCIENCE, Issue 10 2000Maddalena Collini Abstract The fluorescence time decay parameters of the ,-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye,Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge. [source] Tryptophanyl fluorescence lifetime distribution of hyperthermophilic ,-glycosidase from molecular dynamics simulation: A comparison with the experimental dataPROTEIN SCIENCE, Issue 9 2000Ettore Bismuto Abstract A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for ,-glycosidase from the hyperthermophilic bacterium Solfolobus sulfataricus (S,gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of (S,gly) results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71,79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern,Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of S,gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition S,lgy evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results. [source] Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: Fluorescence studyBIOPOLYMERS, Issue 5 2002R. F. Turchiello Abstract The peptide hormone bradykinin (BK) (Arg1 -Pro2 -Pro3 -Gly4 -Phe5 -Ser6 -Pro7 -Phe8 -Arg9) and its shorter homolog BK1,5 (Arg1 -Pro2 -Pro3 -Gly4 -Phe5) were labeled with the extrinsic fluorescent probe ortho -aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH2 and Abz-BK1,5 -NH2). The fragment des-Arg9 -BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg9 -BK-DAP(Abz)-NH2. The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide,lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy,Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK1,5 -NH2 presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 336,346, 2002 [source] Measurement of time-resolved autofluorescenceACTA OPHTHALMOLOGICA, Issue 2008D SCHWEITZER Purpose Functional alterations are first signs of reversible pathologic processes. Whereas microcirculation studies metabolism globally, autofluorescence of endogenous fluorophores has the potential for description of cellular basic processes. Therefore, a discrimination of fluorophores is required in the tissue. Methods Besides excitation and emission spectra, the fluorescence lifetime after short-time excitation is a promising substance-specific mark. Using the opto-mechanical system of a HRA II (Heidelberg Engineering), a fluorescence lifetime mapper was developed. Picosecond pulse-lasers (448nm, 468nm, 100ps FWHM, 80MHz) can be used for excitation and the emission will be detected in 2 spectral ranges (490-560nm, 560-700nm). The dynamic fluorescence will be detected in time-correlated single photon counting (SPC 150, Becker/Hickl, Berlin). An on line image registration is realised by simultaneously detected infrared images during measuring time. Approximating the fluorescence decay by 3-exponential model function, images (lifetime and amplitudes), histograms, and cluster diagrams can be calculated for interpretation. Results Examples are given for healthy subjects, AMD patients (non-exudative, exudative, geographic atrophy), diabetic retinopathy, and oedema. Measurements of excitation and emission spectra as well as lifetimes are performed of expected substances and of anatomical ocular structures for comparison. Conclusion Fluorescence lifetime measurement at the eye is a new method for evaluation of functional metabolic state. [source] Supramolecular Architectures by Fullerene-Bridged Bis(permethyl-,-cyclodextrin)s with PorphyrinsCHEMISTRY - A EUROPEAN JOURNAL, Issue 42 2009Ying-Ming Zhang Abstract The Hirsch,Bingel reaction of bis{4-methyl[1,2,3]triazolyl}malonic ester-bridged bis(permethyl-,-cyclodextrin) 1 with C60 has led to the formation of a new fullerene-bridged bis(permethyl-,-cyclodextrin) 2, which has been comprehensively characterized by NMR spectroscopy, MALDI-MS, and elemental analysis. Taking advantage of the high affinity between 2 and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (3) or [5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato]zinc(II) (4), linear supramolecular architectures with a width of about 2,nm and a length ranging from hundreds of nanometers to micron dimension were conveniently constructed and fully investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM). Significantly, the photoinduced electron-transfer (PET) process between porphyrin and C60 moieties takes place within the 2,3 and 2,4 supramolecular architectures under light irradiation, leading to the highly efficient quenching of the porphyrin fluorescence. The PET process and the charge-separated state were investigated by means of fluorescence spectroscopy, fluorescence decay, cyclic voltammetry, and nanosecond transient absorption measurements. [source] Fluorescence and photoisomerization studies of p -nitrophenyl-substituted ethenylindolesJOURNAL OF PHYSICAL ORGANIC CHEMISTRY, Issue 1 2006Anil K. Singh Abstract The synthesis, electronic absorption, fluorescence (,f, ,ex, ,f, ,f) and photoisomerization (,t,c, photostationary state composition) properties of 3-(4-nitrophenylethenyl- E)-NH-indole (1), 3-(4-nitrophenylethenyl- E)- N -ethylindole (2) and 3-(4-nitrophenyl ethenyl- E)- N -benzenesulfonylindole (3) in organic solvents of varying polarity are reported. The absorption maximum of these compounds undergoes a moderate red shift with increasing solvent polarity. However, the fluorescence maximum becomes highly red shifted with increasing solvent polarity. Whereas 1 and 2 show broad fluorescence bands, 3 exhibits dual fluorescence. Further, 1 and 2 fluoresce much more efficiently than 3. Correlation of the Stokes shift with solvent polarity parameters such as ,f and ET(30) and excited-state dipole moment indicate a highly polar excited state for 1,3. Time-resolved fluorescence studies show that the fluorescence decays are single- and multi-exponential type, depending on the solvent polarity. Further, 1 and 2 do not show photoisomerization on irradiation. However, 3 is photoactive and shows efficient photoisomerization in non-polar heptane. The sensitivity (,) of the photoreaction is determined in various solvent in terms of the Hammett plot, which showed that the excited states involved are electron deficient in nature and consequently stabilized more by an electron sufficient polar solvent and electron donating substituent. These results led us to suggest the existence of three types of excited states, namely the locally excited state, the intramolecular charge-transfer excited state and the conformationally relaxed intramolecular charge-transfer excited state in the photoprocesses of these compounds. Copyright © 2005 John Wiley & Sons, Ltd. [source] Conformational Relaxation of p -Phenylenevinylene Trimers in Solution Studied by Picosecond Time-Resolved FluorescenceCHEMPHYSCHEM, Issue 18 2007Roberto E. Di Paolo Dr. Abstract Two p -phenylenevinylene (PV) trimers, containing 3,-methylbutyloxyl (in MBOPV3) and 2,-ethylhexyloxyl (in EHOPV3) side chains, are used as model compounds of PV-based conjugated polymers (PPV) with the purpose of clarifying the origin of fast (picosecond time) components observed in the fluorescence decays of poly[2-methoxy-5-(2,-ethylhexyloxy)- p -phenylenevinylene] (MEH-PPV). The fluorescence decays of MBOPV3 and EHOPV3 reveal the presence of similar fast components, which are assigned to excited-state conformational relaxation of the initial population of non-planar trimer conformers to lower-energy, more planar conformers. The rate constant of conformational relaxation kCR is dependent on solvent viscosity and temperature, according to the empirical relationship kCR=a,o,,,exp(,,E,/RT), where a,o,, is the frequency factor, ,o is the pre-exponential coefficient of viscosity, E, is the activation energy of viscous flow. The empirical parameter ,, relating the solvent microscopic friction involved in the conformational change to the macroscopic solvent friction (,=1), depends on the side chain. The fast component in the fluorescence decays of MEH-PPV polymers (PPVs), is assigned to resonance energy transfer from short to longer polymer segments. The present results call for revising this assignment/interpretation to account for the occurrence of conformational relaxation, concurrently with energy transfer, in PPVs. [source] Ultrafast Photoisomerization of Photoactive Yellow Protein Chromophore Analogues in Solution: Influence of the Protonation StateCHEMPHYSCHEM, Issue 8 2006Agathe Espagne Dr. Abstract We investigate solvent viscosity and polarity effects on the photoisomerization of the protonated and deprotonated forms of two analogues of the photoactive yellow protein (PYP) chromophore. These are trans- p -hydroxybenzylidene acetone and trans- p -hydroxyphenyl cinnamate, studied in solutions of different polarity and viscosity at room temperature, by means of femtosecond fluorescence up-conversion. The fluorescence lifetimes of the protonated forms are found to be barely sensitive to solvent viscosity, and to increase with increasing solvent polarity. In contrast, the fluorescence decays of the deprotonated forms are significantly slowed down in viscous media and accelerated in polar solvents. These results elucidate the dramatic influence of the protonation state of the PYP chromophore analogues on their photoinduced dynamics. The viscosity and polarity effects are, respectively, interpreted in terms of different isomerization coordinates and charge redistribution in S1. A trans-to-cis isomerization mechanism involving mainly the ethylenic double-bond torsion and/or solvation is proposed for the anionic forms, whereas "concerted" intramolecular motions are proposed for the neutral forms. [source] | |||||||||||||