Polystyrene Microspheres in Tissue-Simulating Phantoms Can Collisionally Quench Fluorescence

Tissue-simulating phantoms that replicate intrinsic optical properties in a controlled manner are useful for quantitative studies of photon transport in turbid biological media. In such phantoms, polystyrene microspheres are often used to simulate tissue optical scattering. Here, we report that using polystyrene microspheres in fluorescent tissue-simulating phantoms can reduce fluorophore quantum yield via collisional quenching. Fluorescence lifetime spectroscopy was employed to characterize quenching in phantoms consisting of a fluorescein dye and polystyrene microspheres (scattering coefficients _s 100-600cm^–1). For this range of tissue-simulating phantoms, analysis using the Stern-Volmer equation revealed that collisional quenching by polystyrene microspheres accounted for a decrease in fluorescence intensity of 6-17% relative to the intrinsic intensity value when no microspheres (quenchers) were present. The intensity decrease from quenching is independent of additional, anticipated losses arising from optical scattering associated with the microspheres. These results suggest that quantitative fluorescence measurements in studies employing such phantoms may be influenced by collisional quenching.     

Spectral-luminescent properties of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and malate dehydrogenase complexes

Spectral confirmation of the formation of stable equilibrium complexes (association constant K _as = 210⁶ M^–1) of the Krebs cycle enzyme — malate dehydrogenase (MDH) — and one of the promising photodynamic sensitizers — chlorin e ₆ — have been obtained. It is shown that the incorporation of dye molecules into the protein globule of dimeric MDH (each subunit of which contains 4 tryptophan amino acid residues, each binding one molecule of chlorin e ₆) is accompanied by quenching of the tryptophan fluorescence of the enzyme. However, despite the overlapping of the fluorescence spectra of tryptophanyls of MDH and the absorption spectrum of chlorin e ₆, the fluorescence quenching observed is not caused by singlet-singlet inductive-resonant transfer of energy from the donor to the acceptor. The conclusion has been drawn that the reason for the absence of energy transfer from tryptophanyls to the dye is the more effective intertryptophan migration of energy to one of the most longwave amino acid residues, the quenching of the luminescence of which occurs due to the reversible photoinduced transfer of an electron to chlorin e ₆ (formation of a complex with charge transfer). The formation of a complex with charge transfer between chlorin e ₆ (when it is excited) and one of the amino acid residues of the enzyme that contact with the dye at its binding site on the protein molecule is also the most noncontradictory explanation of the observed (when bound with MDH) decrease in the quantum yield of fluorescence of chlorin e ₆ upon increase in the duration of its quenching. The question of the ability of MDH to act as one of the most sensitive targets responsible for the disturbance of mitochondrial functions and initiation of the apoptosis of tumor cells in the process of photodynamic therapy is discussed.Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 71, No. 6, pp. 749–758, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Enhancement of uranyl fluorescence using trimesic acid: Ligand sensitization and co-fluorescence

Trimesic acid (TMA) was shown to sensitize and enhance uranyl fluorescence in aqueous medium, with the enhancement being a maximum at pH 5.0. Fluorescence spectra and lifetime data together suggest that TMA complexes with uranyl . The fluorescence of UO₂2+ in its acid complex is further enhanced by more than two orders of magnitude following the addition of Y³⁺; a process referred to as co-fluorescence, leading to the possibility of detecting uranium at sub ng/mL level. The present study demonstrates, for the first time, fluorescence enhancement of the uranyl species due to co-fluorescence.     

Selective Fluorescent Detection of Aspartic Acid and Glutamic Acid Employing Dansyl Hydrazine Dextran Conjugate

Highly water soluble polymer (DD) was prepared and evaluated for its fluorescence response towards various amino acids. The polymer consists of dansyl hydrazine unit conjugated into dextran template. The conjugation enhances higher water solubility of dansyl hydrazine moiety. Of screened amino acids, DD exhibited selective fluorescence quenching in the presence of aspartic acid (Asp) and glutamic acid (Glu). A plot of fluorescence intensity change of DD against the concentration of corresponding amino acids gave a good linear relationship in the range of 1?×?10^?4 M to 25?×?10^?3 M. This establishes DD as a potential polymeric sensor for selective sensing of Asp and Glu.

Spectroscopic investigation of the interaction between thiourea-zinc complex and serum albumin

The interaction between 1-Zn (N-p-(dimethylamino)benzamido-N′-phenylthiourea-zinc) complex and serum albumins was studied. In the presence of proteins such as BSA or HSA, the fluorescence spectrum of 1 did not change. However, the fluorescence intensity of its zinc complex (1-Zn) was greatly enhanced. It was ascribed to the fact that zinc ion promoted the interaction between 1 and proteins. Therefore, it was concluded that zinc ion could facilitate bioactivity of thiourea derivative drugs. Energy transfer occurred between 1-Zn and the proteins, which led to decrease of proteins’ emission and increase of 1-Zn’s emission. The fluorescence quenching of serum albumins by 1-Zn was considered as a static quenching process. The binding constants between 1-Zn and serum albumins were estimated as 1.02×10¹² mol⁻¹ L for BSA and 1.32×10¹⁰ mol⁻¹ L for HSA, respectively, and the number of binding sites was 2 for both. The effect of 1-Zn on the conformation of serum albumins was further investigated using synchronous fluorescence spectrometry and the results implied that tyrosine residues of proteins were closer to 1-Zn than tryptophan residues.     

Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This lifetime quenching can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.Abbreviations PEG poly(ethylene glycol) - Na₂EDTA ethyiene-diamine-tetraacedic acid, disodium salt - LUV large, unilamellar vesicles made by rapid extrusion technique - DPH 1,6-diphenyl-trans-1,3,5-hexatriene - DPHpPC 1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine - DPPC 1,2-dipalmitoyl-3-sn-phosphatidylcholine - PA palmitic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE - Rh-PE N-(lissamine Rhodamine B sulfoyl)-PE - R₁₈ octadecyl Rhodamine B chloride - ANTS 1-aminonaphthalene-3,6,8-trisulfonic acid - DPX N,N-p-xylylene-bis(pyradinium bromide)     

Rotamer-specific fluorescence quenching in tyrosinamide: Dynamic and static interactions

We have examined the environments of the three phenol rotamers about the C–C bond in tyrosinamide by fluorescence quenching. Steady-state acrylamide quenching yields a nonlinear stern-Volmer plot. With three distinct emitting species and no other information about the system, it is impossible to analyze the data due to the number of variables which have to be determined. We therefore reduced the number of variables by independently determining the fractional intensity and dynamic quenching constant for each rotamer through time-resolved fluorescence quenching studies. These parameters were then used to analyze the steady-state data for any contribution of static quenching. We conclude that the nonlinear Stern-Volmer plot for the quenching of tyrosinamide by acrylamide is a consequence of each rotamer having a distinct dynamic quenching constant and the presence of static quenching. The static quenching can be represented by either the sphere-of-action model involving two of the three rotamers or the ground-state complex model involving all three rotamers.     

Tryptophan phosphorescence of ribonuclease T1 as a probe of protein flexibility

The phosphorescence properties of Trp-59 of ribonuclease T₁ fromAspergillus oryzae were monitored as a function of temperature, pH, salt concentration, and complex formation with substrate analogues and, also, in the presence of glycerol as viscogenic cosolvent. The results establish a rough correlation between the internal flexibility of the macromolecule, as derived from the triplet lifetime, and its stability (G orT _m ) toward unfolding. Below 10°C or in 70% glycerol the triplet probe distinguishes at least two gross conformations for the protein, which are characterized by a large difference in phosphorescence lifetime. It is pointed out that such structural heterogeneity does not correspond with the heterogeneity inferred from fluorescence decays and acrylamide quenching rates. Further, implications of the phosphorescence data with regard to the interpretation of acrylamide quenching of fluorescence are discussed.     

Determination of Copper(II) Ion Concentration by Lifetime Measurements of Green Fluorescent Protein

The understanding of cellular processes and functions and the elucidation of their physiological mechanisms is an important aim in the life sciences. One important aspect is the uptake and the release of essential substances as well as their interactions with the cellular environment. As green fluorescent protein (GFP) can be genetically encoded in cells it can be used as an internal sensor giving a deeper insight into biochemical pathways. Here we report that the presence of copper(II) ions leads to a decrease of the fluorescence lifetime (τ _fl) of GFP and provide evidence for Förster resonance energy transfer (FRET) as the responsible quenching mechanism. We identify the His₆-tag as the responsible binding site for Cu^2?+? with a dissociation constant K _d ?=?9 ±2 μM and a Förster radius R ₀?=?2.1 ±0.1 nm. The extent of the lifetime quenching depends on [Cu^2?+?] which is comprehended by a mathematical titration model. We envision that Cu^2?+? can be quantified noninvasively and in real-time by measuring τ _fl of GFP.     

Dipyrrylmetheneboron Difluorides as Labels in Two-Photon Excited Fluorometry. Part II–Nucleic Acid Hybridization Assays

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF₂ labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia^™ TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF₂ labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.     

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

The anomalous temperature dependence of the sound velocity in water

The quantum yields of naphthalene vapour fluorescence at 225°c and of phenanthrene vapour fluorescence at 365°c excited by the Hg 313 μ line are independent of concentration up to 0·014 and 0·007 moles/l. respectively; this is attributed to fast dissociation relaxation of the excimer at these temperatures.

The fluorescence of naphthacene vapour excited at temperatures of 355–435°c by the group of Hg lines at 365 μ decreases with increasing pressure at pressures below that at which absorption of the incident radiation is virtually complete, and is attributed to a combination of self-quenching and reabsorption of fluorescence. An analysis of the data for the limiting cases of complete and negligible fluorescence reabsorption provides upper and lower limits for self-quenching constant which are consistent with unit collisional quenching efficiency and a lifetime of 5·3 ± 2·2 + 10^-9 sec at 355°c.     

On the luminescent behaviour of europium in yttrium oxide and yttrium vanadate hosts

Europium-doped Y₂O₃ and YVO₄ were excited by ultraviolet and 10 kV electrons to give the red emission of Eu³⁺. Increase in the fluorescence output with temperature under uv excitation results from an increased absorption and a more efficient energy transfer to the Eu³⁺ ions from charge-transfer states involving the Y-O and V-O componensts of the lattices. The absence of thermal quenching of fluorescence for (Y₂O₃Eu) is attributed to the high energy of its charge-transfer states which forbids the⁵ D ₀ state to come into thermal contact with them. Complete quenching would occur above 2000 K as predicted from an estimated activation energy of 24 417 cm^–1. Quenching of cathodoluminescence of (YVO₄Eu) commences at 150 K due to the lower energy of its charge-transfer states. The experimentally-deduced temperature for complete quenching of cathodoluminescence for (YVO₄Eu) is lower than that predicted from an estimated thermal activation energy of 14 217 cm^–1; the difference being attributed to localized heating effects induced by electron bombardment. It is suggested that europium ions do not take part in thermoluminescence processes. Electron-hole recombinations occur at host sites to give the observed glow peaks which have been ascribed to traps produced by lattice defects and uncontrollable impurities in the undoped hosts.     

铕螯合物的制备及光谱性质研究

稀土螯合物的制备是均相时间分辨荧光免疫分析中的关键部分,为了合成理想的稀土螯合物,以2,6-二(溴甲基)吡啶-3,5-二甲酸二乙酯为原料,首先优化合成了Li+⊂2,6-{N,N’,N,N’-[二(2,2’-联吡啶-6,6’-二甲基)]二(氨甲基)}-吡啶-二羧酸乙酯,使其产率明显提高。进一步选择乙腈和甲醇两种反应体系合成铕螯合物,并比较了不同反应体系下合成的铕螯合物的光谱性质。研究表明,乙腈和甲醇两种反应体系所得铕螯合物的激发光谱(最大激发波长为310 nm)、发射光谱(最大发射波长为616 nm)、量子产率基本相同,荧光强度在10-8～10-5 mol·L-1范围内与Eu3+浓度均成线性,相关系数分别为0.993 73和0.986 65,两种铕螯合物(c=2.5×10-5 mol·L-1)的荧光强度略有差异,荧光寿命分别为825和830 μs。因此,两种反应体系所得铕螯合物具有斯托克斯位移大、荧光强度强以及荧光寿命长等优点,并且此种穴状螯合剂结构中的吡啶-2,2-联吡啶可保护铕离子免受其他物质的干扰,是理想的稀土螯合物,可用于蛋白质、核酸等生物分子的标记。本研究不仅拓展了合成新型稀土螯合物的方法,而且为进一步建立均相时间分辨荧光免疫分析奠定了基础。     

Optical transitions of Dy in tellurite glass: observation of upconversion

Dy³⁺ doped tellurite glasses were prepared and its optical absorption, fluorescence and upconversion have been studied. The absorption data has been used to calculate the Judd-Ofelt parameters, oscillator strengths along with transition probability, branching ratio, etc. The lifetime of the level is found to be 0.66 ms for 1.0 mol% of Dy³⁺ doped in tellurite glass. NIR to visible upconversion has been observed at room temperature. The increase in upconversion luminescence at lower temperature indicates that upconversion is not due to a thermally activated process. The quantum efficiency of the transition is found to be ∼0.17 and the upconversion efficiency is ∼0.022. The strong dependence of the upconversion efficiency on the Dy³⁺ concentration reveals that the upconversion is due to energy transfer interaction.     

Wide- and narrow-band saturated fluorescence measurements of hydroxyl concentration in premixed flames from 1 bar to 10 bar

We have studied the use of wide-band detection in conjunction with saturation of a rovibronic transition of OH within itsA ^{2 +}–X ²(0,0) band. For wide-band detection, in which fluorescence is detected from the entire excited rotational manifold, the fluorescence yield is sensitive to collisions in two ways. First, it is sensitive to the ratio of rate coefficients describing rotational energy transfer and electronic quenching; this ratio determines the number of neighboring rotational levels that are populated during the laser pulse. Second, the fluorescence yield can vary with the total collisional rate coefficient; only after a sufficient number of collisions, corresponding to 2.5 ns in an atmospheric flame, does the rotational manifold reach steady state. We also compare measurements employing wide-band (detecting theR ₁ andR ₂ branches) and narrow-band (detecting a single transition) saturated fluorescence of OH. Over a wide range of conditions — obtained by varying the equivalence ratio, temperature, N₂ dilution, and pressure — the wide- and narrow-band fluorescence techniques compare well. Given this good agreement, wide-band saturated fluorescence could be especially useful for analyzing atmospheric flames with XeCl-excimer lasers; one can potentially obtain 2—D images of OH which have a high signal-to-noise ratio and a reduced sensitivity to laser irradiance and quenching.     

Fluorimetric Determination of Thyroxine Hormone with Eu(III)-(Pyridine-2,6-Dicarboxylate) Tris Complex

We describe a method for the determination of thyroxine (Thy) using its quenching effect on Eu(PDA)₃ ^3- tris complex fluorescence. The relative fluorescence intensities are measured at fixed _exc = 282 nm, _em = 615 nm by titrating the metal complex with Thy in piperazine buffer solution at pH 6.5. Data indicated an associative type of reaction of two molecules valid between 0.0 < R < 1.0, R being the mole ratio of Eu(PDA)₃ ^3- to Thy. Over this ratio and up to (R 1.0) collisional quenching of Eu(PDA)₃ ^3- complex ion emission is seen as a result of heavy atom effect, intermolecular energy transfer playing the main role. This is also confirmed by the Stern-Volmer equation. In optimized experimental conditions, the L- form of Thy is determined in a range of 15.5–551.6 g/ml (2.0 × 10^–5 – 7.1 × 10^–4 M) with relative error of ±1.17%.     

A Rhodamine-Based Off�COn Fluorescent Chemosensor for Selectively Sensing Cu(II) in Aqueous Solution

A novel chromogenic and fluorogenic chemosensor RhB-pMOSal comprising a rhodamine fluorophore and a salicylaldehyde receptor being connected by an iminohydrazine link was synthesized and fully characterized. Its sensing behavior toward various metal ions in neutral aqueous solution was investigated by absorption and fluorescence spectroscopy. RhB-pMOSal exhibited a reversible and sensitive ??turn-on?? response of absorption and fluorescence toward Cu²⁺ in aqueous acetonitrile solution. Approximate 65 and 6-fold enhancement in the absorbance at 556 nm and fluorescence intensity at 573 nm were estimated when equivalent Cu²⁺ was added to the RhB-pMOSal solution. Under the same conditions, RhB-pMOSal displayed more sensitive than a reported analogue RhB-Sal to Cu²⁺ ion. The competition experiments for Cu²⁺ mixed with common metal ions exhibited no obvious change in absorption and emission except Cr³⁺ ion that can induce the fluorescence quenching of RhB-pMOSal to some extent.     

Fluorescence and attenuation properties of Er3+ -doped phosphate-glass fibers and efficient infrared-to-visible up-conversion

Fluorescence spectra of phosphate glasses with different erbium doping have been measured. The flourescence intensity reaches its maximum at an Er³⁺-doping concentration of 0.75 mol%. When the Er³⁺ doping exceeds 0.75 mol%, the fluorescence intensity decreases due to concentration quenching. The attenuation at 1.53 µm of the fiber is 12.8 db/m. The fluorescence up-conversion of 1.064 µm Nd:YAG laser pulses into intense green 547 and 667 nm light in the fiber has been measured. The fluorescence output power of green (547 nm) and red (667 nm) light is 178 and 42 W, respectively with an excitation power of 1 W. The two signals are referred to as⁴ S _3/2 ⁴ I _{1 5/2} and⁴ F _9/2 ⁴ I _{1 5/2} transitions through two-photon absorption fluorescence.     

Two-dimensional quenching lifetime measurement of OH:A 2 Σ +(υ′ = 1) and NO:A 2 Σ(υ′ = 0) in atmospheric-pressure flames

Planar laser-induced fluorescence is currently widely applied to research on combustion. However, conventional approaches for semi-quantitative measurement could not provide the satisfactory solution and the problem due to collisional quenching remains to be solved. This paper describes the two-dimensional (2D) quenching-time-constant measurement of OH:A ^{2 +}( = 1) and NO:A ² ( = 0) in flame cross sections at atmospheric pressure. These measurements involve 1–2 ns decay time of the excited state using a high-speed image intensifier and a tunable laser with a pulse duration of 3 ns at FWHM. The correlation factors of the exponential fits for the fluorescence decays after the laser pulse were larger than 0.999 in all experiments presented in this study. Furthermore, the measured Stern-Volmer plots of the quenching rate at 1.0, 1/2, 1/3, and 1/4 atm pressure using the same experimental apparatus was confirmed to have a linear relationship for both OH and NO, showing that the 2D decay-time measurements of 1–2 ns have been successful. For NO:A ² ( = 0), the obtained quenching rates inside the inner cone and in the outer flame in the NO-seeded methane-air Bunsen flame were 8.7 × 10⁸ and 7.8 × 10⁸ s^–1, respectively, and for OH:A ² ( = 1), the obtained quenching rate mapping in the outer flame was around 5.6 × 10⁸ s^–1 in the methane-air flame.