LIGHT QUENCHING OF FLUORESCENCE: A NEW METHOD TO CONTROL THE EXCITED STATE LIFETIME AND ORIENTATION OF FLUOROPHORES

Abstract Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modem high-repetition rate lasers, a phenomenon we call “light quenching.” In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.     

Employing the fluorescence anisotropy and quenching kinetics of tryptophan to hunt for residual structures in denatured proteins

Residual structures in denatured proteins have acquired importance in recent years owing to their role as protein-folding initiation sites. Locating these structures in proteins has proved quite formidable, requiring techniques like NMR. Here in this report, we take advantage of the ubiquitous presence of tryptophan residues in residual structures to hunt for their presence using steady-state fluorescence spectroscopy. The surface accessibility and rotational dynamics of tryptophan in putative residual structures among ten different proteins, namely glucagon, melittin, subtilisin carlsberg, myelin basic protein, ribonuclease T₁, human serum albumin, barstar mutant, bovine serum albumin, lysozyme and Trp-Met-Asp-Phe-NH₂ peptide, was studied using steady state fluorescence quenching and anisotropy, respectively. Five proteins, namely ribonuclease T₁, bovine serum albumin, melittin, barstar and hen egg white lysozyme appear likely to possess tryptophan(s) in hydrophobic clusters based on their reduced bimolecular quenching rates and higher steady-state anisotropy in proportion to their chain length. We also show that the fluorescence emission maximum of tryptophan is insensitive to the presence of residual structures.     

Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation

Topotecan is an antitumor agent with activity against a variety of cancers. We examined the steady-state and time-resolved fluorescence spectral properties of topotecan with one- and two-photon excitation. Topotecan was found to display a high two-photon cross section near 20 GM for wavelengths within the fundamental output of a Ti:sapphire laser, 800-880 nm. In frozen solution the anisotropies of topotecan are near the theoretical maxima for one-photon and two-photon excitation with colinear electronic transitions. The intensity and anisotropy decays of topotecan fluorescence were found to be homogeneous (single exponentials) in phosphate-buffered saline and propylene glycol. The steady-state and time-resolved data indicate that topotecan binds to a double-helical DNA oligomer d(AT)10 resulting in increased anisotropies and multiexponential intensity and anisotropy decays. Subnanosecond components in the anisotropy decay of the DNA-topotecan complex suggest loose binding of the drug to DNA. Loose binding of topotecan to DNA is also revealed by accessibility of topotecan to collisional quenching by iodide.     

Compartmental modeling of the fluorescence anisotropy decay of a cylindrically symmetric brownian rotor: Identifiability analysis.

We present the results of the deterministic identifiability analysis based on similarity transformation for models of one-state excited-state events of cylindrically symmetric rotors in isotropic environments undergoing rotational diffusion described by Brownian reorientation. Such an analysis on error-free time-resolved fluorescence (anisotropy) data can reveal whether the parameters of the considered model can be determined. The fluorescence delta-response functions I(parallel)(t) and I(perpendicular)(t), for fluorescence polarized respectively parallel and perpendicular to the electric vector of linearly polarized excitation, are used to construct, in convenient matrix form, expressions of the sum S(t) = I(parallel)(t) + 2I(perpendicular)(t), the difference D(t) = I(parallel)(t) - I(perpendicular)(t), and the time-resolved fluorescence anisotropy r(t) = D(t)/S(t). The identifiability analysis of r(t) demonstrates that the rotational diffusion coefficients D(parallel) and D(perpendicular) for rotation respectively about and perpendicular to the symmetry axis can be uniquely resolved. However, the polar and azimuthal angles defining the absorption and emission transition moments in the molecular reference frame are not individually identifiable. Nevertheless, the difference between the polar angles of these transition moments is uniquely determined.     

Fluorimetric study of interaction of merbromin with trypsin

Interaction of merbromin with trypsin is of bovine origin has been studied by monitoring the absorption steady-state and time-resolved fluorescence spectral properties of the dye. Studies have been done in media of varying pH at different trypsin concentrations. It has been observed that trypsin brings about a quenching of fluorescence of the dye. The quenching is static in nature and the equilibrium constant of dye-trypsin interaction in the ground-state has been determined from quenching studies. Steady-state anisotropy of the dye increases in presence of trypsin in the medium. Values of micro-viscosity in the vicinity of the fluorophore in media containing trypsin have been determined from measurements of fluorescence anisotropy. Time-resolved fluorescence studies indicate the existence of two decaying states for the dye. The fractional contribution to the time-resolved decay changes with pH. The average lifetime, however, does not depend on the concentration of trypsin.     

Chiral recognition of binaphthyl derivatives using electrokinetic chromatography and steady-state fluorescence anisotropy: effect of temperature

The effect of temperature on the chiral recognition of binaphthyl derivatives in the presence of poly sodium N-undecanoyl-LL-leucyl-leucinate (poly LL-SULL) is examined using electrokinetic chromatography (EKC) and steady-state fluorescence anisotropy. An examination of the effect of temperature suggests that the chiral recognition of 1,1'-binaphthyl-2,2'-diol enantiomers improves with increasing temperature, whereas lower temperatures resulted in better enantiosolectivity in the case of 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate enantiomers. In addition, steady-state fluorescence anisotropy results show that the anisotropy of the two enantiomers are different when complexed to poly-(LL) SULL. As would be expected, the enantiomer that binds stronger to the chiral pseudostationary phase, as evidenced by EKC experiments, had higher anisotropy values. The results of this study suggest that steady-state fluorescence anisotropy can be used to gain further insight into chiral recognition.     

一种新型pH荧光探针：中位-吡啶取代的硼二吡咯亚甲基(BDP)染料的合成、结构与光谱性质研究

本文报道了一种新型中位-吡啶取代的硼-二吡咯亚甲基(BDP)染料的合成与晶体结构表征,发现中位-吡啶环与因达省平面几乎正交,其二面角为88.2°。同时对它的吸收和稳态荧光性质研究表明：当溶液的酸性增强时,化合物的荧光减弱,pK_a为2.24。该化合物在可见光激发下,可以作为比较灵敏的pH荧光探针。     

A Paramagnetic Compass Based on Lanthanide Metal-Organic Framework

Macroscopic compass-like magnetic alignment at low magnetic fields is natural for ferromagnetic materials but is seldomly observed in paramagnetic materials. Herein, we report a “paramagnetic compass” that magnetically aligns under ∼mT fields based on the single-crystalline framework constructed by lanthanide ions and organic ligands (Ln-MOF). The magnetic alignment is attributed to the Ln-MOF's strong macroscopic anisotropy, where the highly-ordered structure allows the Ln-ions’ molecular anisotropy to be summed according to the crystal symmetry. In tetragonal Ln-MOFs, the alignment is either parallel or perpendicular to the field depending on the easiest axis of the molecular anisotropy. Reversible switching between the two alignments is realized upon the removal and re-adsorption of solvent molecules filled in the framework. When the crystal symmetry is lowered in monoclinic Ln-MOFs, the alignments become even inclined (47°-66°) to the field. These fascinating properties of Ln-MOFs would encourage further explorations of framework materials containing paramagnetic centers.     

Determination of Fluorescence Polarization and Absorption Anisotropy in Molecular Complexes Having Threefold Rotational Symmetry

Abstract— The current work concerns investigation of the polarization properties of complex molecular ensembles exhibiting threefold (C₃) rotational symmetry, particularly with regard to the interplay between their structure and dynamics of internal energy transfer. We assume that the molecules or chromophores in such complexes possess strongly overlapped spectra both for absorption and fluorescence. Such trimeric structures are widely found in biological preparations, as for example the trimer of C-phycocyanin (C-PC). Higher order aggregates, e.g. hex-amers and three-hexamer rods, are also investigated and compared with the trimer case. The theory addresses both steady-state and 8-pulse excitation and establishes some links between them. Monochromophoric, bichro-mophoric and trichromophoric molecular complexes are individually examined. For steady-state excitation, analytical formulas are reported for the degree of fluorescence polarization and absorption anisotropy. It is shown that the polarization is dependent on the chromophore inclination relative to the symmetry axis, the relative efficiencies of absorption and fluorescence by chromophores of different spectral types, and the rates of energy equilibration. To assess the validity of the theory, it has been applied to C-PC aggregates. Here it was found that different C-PC aggregates provide practically identical polarization response. For S-pulse excitation we give analytical formulas for determination of the fluorescence depolarization, and also the depolarization associated with absorption recovery, both for a monochromophoric trimer and some particular cases of bichromophoric trimer. More complicated systems are analyzed by computer modeling. Thus it transpires that the initial polarization anisotropy r(t = 0) takes the value 0.4 for all considered aggregates; the long-time limit r(t →∞) has about the same value as is associated with steady-state excitation. We also show that with steady-state excitation the degree of fluorescence polarization is practically equal for various C₃ aggregates of C-PC, and that the major factor determining the polarization is the chromophore orientation relative to the symmetry axis.     

基于第一性原理计算的铁电材料BaTiO3相关振动光谱指认

通过固相反应烧结法在1400 ℃下烧结4 h合成了BaTiO₃陶瓷, 并用X衍射确定了其为四方晶系. 进行了拉曼谱和红外谱的测量, 并采用洛仑兹函数以及四参数方法分别对上述光谱进行了拟合. 基于第一性原理的计算, 并考虑了横模纵模劈裂, 对拉曼和红外光谱进行了指认. 为了更好地分析振动模式, 所有振动模用群论的对称坐标进行了分解. 在12个光学模中, 仅具有拉曼活性的B₁模式是O4和O5沿着z轴的相对运动. A₁模式和E₁软模式是从立方BaTiO₃相的F_1u模式劈裂出来的, 对于四方相BaTiO₃的铁电性有着重要作用, 其体现在A₁⁽¹⁾模式造成了铁电z轴的极化, E₁模式导致了大介电常数. 这两个模式都可以看成是Ti 原子相对于O₆八面体笼子沿着z轴或者是xy平面的振动.     

The mechanical anisotropy of stratified polyvinyl chloride

The mechanical anisotropy of a material obtained by stratification of oriented polyvinyl chloride (PVC) layers in different ways has been studied. PVC layers, uniaxially oriented by drawing each with a different draw ratio, were stratified in such a way that their draw axes were either parallel or perpendicular. The propagation velocities of ultrasonic pulses were measured in three principal planes defined by draw axes of the stratified material. With an immersion technique, measurements were performed at 2 MHz and 22°C. Experimentally, it was shown that while stratification of layers with draw axes parallel preserves hexagonal symmetry, the stratified material with layers' draw axes perpendicular possesses orthorhombic symmetry. © 1994 John Wiley & Sons, Inc.     

Field quenching of director fluctuations in thin films of nematic liquid crystals

The continuum theory of director fluctuations in nematic liquid crystals is extended to take account of finite sample size which more accurately describes experiments on thin films. The effect of external fields on director fluctuations is considered for two cell geometries: (i) the field parallel to the director for materials of positive susceptibility anisotropy, and (ii) the field perpendicular to the director for materials of negative susceptibility anisotropy. In the latter geometry, quenching of fluctuations leads to induced biaxiality. Comparison with experimental results shows that even allowing for the finite size of the sample, there is still a significant disagreement between theory and experiment, especially from thin samples.     

DISTANCE-DEPENDENT FLUORESCENCE QUENCHING OF TRYPTOPHAN BY ACRYLAMIDE

Abstract We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N -acetyl -L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20°C. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NAT A by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.     

Optical activity of the anisotropic phases of cellulose acetates

In this study, the optical activity of cellulose di and triacetate films with a highly oriented vitrified structure was studied. The systems possess high values of specific optical rotation [α], which is in favor of the formation of a cholesteric mesophase. [α] depends on the sample orientation angle relative to the light polarization vector in the plane perpendicular to the light ray (optical activity anisotropy). This dependence (indicatrix) is irregular and is described by a distorted sinusoid in the Cartesian coordinates. The results of indicatrix decomposition into harmonic components are discussed, with the contributions of the isotropic and anisotropic components (each with the corresponding structural element of a certain symmetry type) being separated. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1605–1615, 2009     

Direct and indirect monitoring of peptide-silica interactions using time-resolved fluorescence anisotropy

The present work extends the application of time-resolved fluorescence anisotropy (TRFA) of a cationic probe rhodamine 6G (R6G) in aqueous Ludox to in situ monitoring of peptide adsorption onto the silica particles. Steady-state anisotropy and TRFA of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE), and N-acetylated Glu-Trp-Glu (Ac-EWE). The results were compared to those obtained by direct observation of peptide adsorption using the steady-state anisotropy of the intrinsic tryptophan residue. Ionic binding of the peptides to Ludox particles produced an increase in the steady-state Trp anisotropy that was dependent on the number of cationic groups present, but the limiting anisotropy values were relatively low, indicating significant rotational freedom of the indole residue in the adsorbed peptides. On the other hand, R6G showed significant decreases in anisotropy in the presence of cationic peptides, consistent with the cationic peptides blocking the adsorption of the dye to the silica surface. Thus, R6G is able to indirectly report on the binding of peptides to Ludox particles. It was noteworthy that, while there were similar trends in the data obtained from steady-state anisotropy and TRFA studies of R6G, the use of steady-state anisotropy to assess binding of peptides overestimated the degree of peptide adsorption relative to the value obtained by TRFA. The study shows that the competitive binding method can be used to assess the binding of various biologically relevant compounds onto silica surfaces and demonstrates the potential of TRFA for probing peptide-silica and protein-silica interactions.     

Directional states of symmetric-top molecules produced by combined static and radiative electric fields

We show that combined electrostatic and radiative fields can greatly amplify the directional properties, such as axis orientation and alignment, of symmetric top molecules. In our computational study, we consider all four symmetry combinations of the prolate and oblate inertia and polarizability tensors, as well as the collinear and perpendicular (or tilted) geometries of the two fields. In, respectively, the collinear or perpendicular fields, the oblate or prolate polarizability interaction due to the radiative field forces the permanent dipole into alignment with the static field. Two mechanisms are found to be responsible for the amplification of the molecules' orientation, which ensues once the static field is turned on: (a) permanent-dipole coupling of the opposite-parity tunneling doublets created by the oblate polarizability interaction in collinear static and radiative fields and (b) hybridization of the opposite parity states via the polarizability interaction and their coupling by the permanent dipole interaction to the collinear or perpendicular static field. In perpendicular fields, the oblate polarizability interaction, along with the loss of cylindrical symmetry, is found to preclude the wrong-way orientation, causing all states to become high-field seeking with respect to the static field. The adiabatic labels of the states in the tilted fields depend on the adiabatic path taken through the parameter space comprised of the permanent and induced-dipole interaction parameters and the tilt angle between the two field vectors.     

非晶Co-Pt合金纳米线有序阵列的制备及其磁学性质

通过直流电沉积方法，以多孔阳极氧化铝(AAO)为模板，在室温下成功制备出一维非晶态Co-Pt合金纳米线有序阵列. SEM和TEM分析表明：纳米线长度均约10 μm，直径35 nm；纳米线在阳极氧化铝模板孔内互相平行. XRD结果表明，制备的纳米线为非晶态结构，经过700 ℃退火处理后则转变为面心立方(FCC)多晶结构. 采用VSM(振动样品磁强计)对退火处理前后样品的矫顽力和剩磁比进行研究，结果表明：当外加磁场与纳米线平行时，非晶态Co-Pt合金纳米线的矫顽力高达1700 Oe，剩磁比为0.83，表现出明显的垂直磁各向异性；而退火处理则使其优秀的磁学性质消失. 退火前后不同的磁学性质源于其不同的微观结构. 非晶态的Co-Pt合金纳米线由于无磁晶各向异性竞争，进而使得由纳米线一维形态引起的形状各向异性起主导作用，使其显示了很好的垂直磁各向异性；而多晶样品由于磁晶各向异性与形状各向异性竞争，导致矫顽力和剩磁比迅速降低.     

Magnetic alignment study of rare-earth-containing liquid crystals

The liquid-crystalline rare-earth complexes of the type [Ln(LH)3(DOS)3]-where Ln is Tb, Dy, Ho, Er, Tm, or Yb; LH is the Schiff base N-octadecyl-4-tetradecyloxysalicylaldimine; and DOS is dodecylsulfate-exhibit a smectic A phase. Because of the presence of rare-earth ions with a large magnetic anisotropy, the smectic A phase of these liquid crystals can be easier aligned in an external magnetic field than smectic A phases of conventional liquid crystals. The magnetic anisotropy of the [Ln(LH)3(DOS)3] complexes was determined by measurement of the temperature-dependence of the magnetic susceptibility using a Faraday balance. The highest value for the magnetic anisotropy was found for the dysprosium(III) complex. The magnetic alignment of these liquid crystals was studied by time-resolved synchrotron small-angle X-ray scattering experiments. Depending on the sign of the magnetic anisotropy, the director of the liquid-crystalline molecules was aligned parallel or perpendicular to the magnetic field lines. A positive value of the magnetic anisotropy (and parallel alignment) was found for the thulium(III) and the ytterbium(III) complexes, whereas a negative value of the magnetic anisotropy (and perpendicular alignment) was observed for the terbium(III) and dysprosium(III) complexes.     

Proton magnetic shielding tensor in liquid water

The nuclear magnetic shielding tensor is a sensitive probe of the local electronic environment, providing information about molecular structure and intermolecular interactions. The magnetic shielding tensor of the water proton has been determined in hexagonal ice, but in liquid water, where the tensor is isotropically averaged by rapid molecular tumbling, only the trace of the tensor has been measured. We report here the first determination of the proton shielding anisotropy in liquid water, which, when combined with chemical shift data, yields the principal shielding components parallel (sigma(parallel)) and perpendicular (sigma(perpendicular)) to the O-H bond. We obtained the shielding anisotropy sigma(parallel)-sigma(perpendicular) by measuring the proton spin relaxation rate as a function of magnetic induction field in a water sample where dipole-dipole couplings are suppressed by H/D isotope dilution. The temperature dependence of the shielding components, determined from 0 to 80 degrees C, reflects vibrational averaging over a distribution of instantaneous hydrogen-bond geometries in the liquid and thus contains unique information about the temperature-dependent structure of liquid water. The temperature dependence of the shielding anisotropy is found to be 4 times stronger than that of the isotropic shielding. We analyze the liquid water shielding components in the light of previous NMR and theoretical results for vapor and ice. We show that a simple two-state model of water structure fails to give a consistent interpretation of the shielding data and we argue that a more detailed analysis is needed that quantitatively relates the shielding components to hydrogen bond geometry.     

The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid membranes under the steady-state conditions

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.