Mechanisms of Fluorescence Quenching in Donor—Acceptor Labeled Antibody—Antigen Conjugates

The cyanine dyes Cy5 and Cy5.5 are presented as a new long wavelength-excitable donor-acceptor dye pair for homogeneous fluoroimmunoassays. The deactivation pathways responsible for the quenching of the fluorescence of the antibody-bound donor are elucidated. Upon binding of the donor dye to the antibodies at low dye/protein ratios, its fluorescence quantum yield rises to unity. Higher dye/protein ratios lead to progressive aggregation of the dyes, which results in quenching of monomer fluorescence due to resonance energy transfer (RET) from the monomers to the nonfluorescent dimers. The dependence of the quenching efficiency on the labeling ratio is described quantitatively by assuming a Poisson distribution of the dyes over the antibodies. The maximum fluorescence intensity per antibody is obtained at a labeling ratio of 4. Upon formation of the antibody-antigen complex, electron transfer and RET to the antigen-bound acceptor dye occur. Steady-state and time-resolved fluorescence measurements reveal that approximately 50% of the donor quenching is due to RET, while the residual quenching effect is caused by the static quenching process.     

Effect of concentration on optical properties of dyes incorporated into polyvinyl alcohol films

The quantum yield, lifetime and anisotropy of fluorescence emitted by molecules of neutral red dye incorporated into polyvinyl polymer films were studied as a function of dye concentration. From the data a mechanism of excitation quenching by dye monomers bound to the polymer matrix is suggested.     

Comparative photophysical and energy transfer studies of C480:C535 binary dye mixture in solid and liquid environments

The spectral behaviour of the individual dyes (C480 and C535) is discussed. The absorption and fluorescence spectral profiles of the dyes, in solid and liquid environments, are identical. Dye doped polymer rods of donor dye (C480) without the acceptor dye (C535), acceptor dye (C535) without the donor dye (C480) and donor dye with acceptor dye at different acceptor concentrations are synthesized. Energy transfer technique is studied, in detail, using these rods. The results obtained are compared with their respective liquid mixtures. Optical gain of the acceptor dye with and without the donor dye is determined experimentally, in both solid and liquid media. The gain of the acceptor with donor increases and then decreases with increase in acceptor concentration. Both solid and liquid media show similar trends. But, the gain coefficient in the solid matrix is less than that in the liquid medium. The photobleaching of the dye doped polymer rod under nitrogen laser excitation is studied.     

Fluorescence decay times of indigo-derivatives measured by means of a synchroscan streak camera

Employing a synchronously pumped, modelocked dye laser for excitation in connection with a commercial, continuously operated streak camera the solvent dependent fluorescence decaytimes of several indigo-derivatives exhibiting a low fluorescence quantum efficiency were determined with a temporal resolution of about 5 ps in order to further elucidate their energy relaxation mechanisms, which are the object of continuous controversy.     

香豆素440/荧光素钠/藏红混合染料体系的光谱研究

通过对C440/Fl.Na/Saf.-T三元混合染料体系的荧光光谱研究,了解到这三种染料之间有较充分的能量转移。又通过对C440/Fl.Na和Fl.Na/Saf.-T两个混合染料体系的荧光寿命测量,讨论了分子间能量转移过程和机理,并做了一些定量的分析。     

Energy migration and transfer between chromophores embedded in Gaussian space

We have used a Monte Carlo method to investigate energy migration and transfer between chromophores embedded in Gaussian space. In using this method we have obtained fluorescence quantum yields, fluorescence depolarization, and the respective decay profiles of donor fluorescence. It was shown that all photophysical observables are dependent upon the number of donor and acceptor chromophores and upon the Förster radii ratio. The latter feature is particularly interesting, and it indicates the existence of correlations between donor and acceptor chromophores. It was shown that the excitation of the donor chromophore at the origin leads to different values of observables, in comparison with an excitation of a randomly selected donor chromophore. The results presented show the importance of the averaging procedures needed to be developed while dealing with specific distribution functions, as, for example, in the case of energy migration and trapping in aromatic polymers and copolymers.     

Fluorescence and Laser Emission from Coumarin-Acridine Orange Mixtures

The sensitized fluorescence and laser emissions of dye mixtures; (I) coumarin 102 (donor) and acridine orange (acceptor) and (II) coumarin 47 (donor) and acridine orange (acceptor) with Hg-lamp and N₂ laser, have been measured as a function of dye concentration and of the pump power (N₂ laser). Acridine orange which does not lase by itself on excitation with N₂ laser, lases efficiently in the presence of 7-amino-coumarins via singlet-singlet energy transfer. Energy transfer rate constants and critical distances have been estimated from fluorescence intensity and lifetime measurements. The performance of energy transfer dye lasers (ETDLs) are discussed in terms of spectral characteristics of the dyes and their penetration depths.     

Enhanced Emission Induced by FRET from a Long-Lifetime, Low Quantum Yield Donor to a Long-Wavelength, High Quantum Yield Acceptor

We report observation of high quantum yield, long-lifetime fluorescence from a red dye BO-PRO-3 excited by resonance energy transfer (RET). The acceptor fluorescence was highly enhanced upon binding to the donor-labeled DNA. A ruthenium complex (Ru) was chosen as a donor in this system because of its long fluorescence lifetime. Both donor and acceptor were non-covalently bound to DNA. Emission from the donor-acceptor system (DA) at wavelengths exceeding 600 nm still preserves the long-lifetime component of the Ru donor, retaining average fluorescence lifetimes in the range of 30–50 ns. Despite the low quantum yield of the Ru donor in the absence of acceptor, its overall quantum yield of the DA pair was increased by energy transfer to the higher quantum yield acceptor BO-PRO-3. The wavelength-integrated intensity of donor and acceptor bound to DNA was many-fold greater than the intensity of the donor and acceptor separately bound to DNA. The origin of this effect is due to an efficient energy transfer from the donor, competing with non-radiative depopulation of the donor excited state. The distinctive features of DA complexes can be used in the development of a new class of engineered luminophores that display both long lifetime and long-wavelength emission. Similar DA complexes can be applied as proximity indicators, exhibiting strong fluorescence of adjacently located donors and acceptors over the relatively weak fluorescence of separated donors and acceptors.     

Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation

Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments. Received: 4 July 2001 / Published online: 23 November 2001     

Thermal lens technique to evaluate the fluorescence quantum yield of a schiff base

The fluorescence spectrum of the schiff base obtained from salicylaldehyde and 2-aminophenol is studied using an argon-ion laser as the excitation source and its fluorescence quantum yield (Q_f) is determined using a thermal lens method. This is a nondestructive technique that gives the absolute value of Q_f without the need for a fluorescence standard. The quantum-yield values are calculated for various concentrations of the solution in chloroform and also for various excitation wavelengths. The value of Q_f is relatively high, and is concentration dependent. The maximum value of Q_f obtained is nearly 0.78. The high value of the fluorescence quantum yield will render the schiff base useful as a fluorescent marker for biological applications. Photostability and gain studies will assess its suitability as a laser dye. PACS 42.55.Mv; 42.62.Cf; 42.62.Fi; 42.70.Hj     

ZnS∶Cu-罗丹明B的荧光共振能量转移性质

为了解决现有的基于量子点荧光共振能量转移体系的生物毒性问题,选用无毒的ZnS∶Cu量子点与罗丹明B构建新型荧光共振能量转移体系。通过共沉淀法成功制备了形貌均一的ZnS∶Cu纳米晶量子点。在此基础上,测试了不同掺杂浓度的ZnS∶Cu量子点及罗丹明B的荧光光谱。然后,通过对ZnS∶Cu量子点的表面修饰构建了以ZnS∶Cu量子点为供体、罗丹明B为受体的荧光共振能量转移体系。实验结果表明:ZnS∶2%Cu量子点的发光光谱与罗丹明B的吸收光谱在481 nm处有较大重合,说明构建荧光共振能量转移的最佳铜掺杂摩尔分数为2%。通过计算发现以ZnS∶2%Cu量子点为供体、罗丹明B为受体的荧光共振能量转移体系的能量转移效率为25.8%。进一步实验结果表明,罗丹明B浓度也能够影响能量转移。     

Spectroscopic and travelling-wave lasing characterisation of tetraphenylbenzidine and di-naphtalenyl-diphenylbenzidine

The organic light emitting diode (OLED) hole transport molecules N,N,N’,N’-tetraphenylbenzidine (TPB, triphenylamine dimer TAD or TPD) and N,N’-bis(2-naphtalenyl)-N,N’-bis(phenylbenzidine) (β-NPB, naphtyl-diphenylamine dimer β-NPD), dissolved in tetrahydrofuran (THF) and as neat film, are characterized by optical absorption and emission spectroscopy. The absorption and stimulated emission cross-section spectra, the fluorescence quantum distributions, fluorescence quantum yields, degrees of fluorescence polarization, and fluorescence lifetimes are determined. The lasing behaviour is studied by picosecond laser pulse excitation (excitation wavelength 347.15 nm, pulse duration 35 ps). The excited-state absorption at the pump laser wavelength is determined by saturable absorption measurement. Low-Q laser oscillation of TPB in THF is achieved by transverse pumping of the dye in a cell. The excited-state absorption of TPB in THF at the laser wavelength is extracted from the laser threshold. In TPB neat films, wave-guided travelling-wave lasing was obtained. No laser action was achieved for β-NPB because of small S₁-S₀ stimulated emission cross-section, and the presence of excited-state absorption in the fluorescence wavelength region. The TPB and β-NPB results are compared with the corresponding spectroscopic and lasing behaviour of the related methyl-substituted triphenylamine dimers, 3-methyl-TPD and 4-methyl-TPD, which are well established OLED hole transport materials.     

Intermolecular energy transfer in mixed laser dyes: photophysical properties of triplet states

Triplet-singlet energy transfer in laser dyes have been studied in EPA at 77K using N₂ laser as an excitation source. Phosphorescence of the donor (D) and the delayed fluorescence of the acceptor (A) and their lifetimes have been measured for coumarin 102 (D)-rhodamine B(A) and 9(10H)-acridone (D)-rhodamine 6G(A) dye systems as a function of acceptor concentration. These data yield energy transfer rate constants of ∼10³ dm³ mol⁻¹ s⁻¹ for the donor acceptor combinations, consistent with the Forster mechanism. The phosphorescence quantum efficiency and other spectral parameters are also reported.     

Effect of cooperative interactions in optical ensembles on the threshold characteristics of lasing in the case of coherent optical pumping

The dependence of the threshold pump energy of dye lasers on the quantum yield of fluorescence of active solutions in the case of laser excitation was studied. The rate of increase in the threshold pump energy was found to be considerably lower than the rate of decrease in quantum yield. The results were interpreted in terms of the development of the cooperative interaction between active centers in the high-intensity coherent pump field.

     

Photophysical properties, laser activity and photoreactivity of a heteroaryl chalcone: A model of solvatochromic fluorophore

The absorption and fluorescence characteristics of 3-(4′-dimethylaminophenyl)-1-(2-thienyl)prop-2-en-1-one (DMATP) have been investigated in different solvents. DMATP dye exhibits a large red shift in both absorption and emission spectra as solvent polarity increases, indicating a large change in the dipole moment of molecules upon excitation due to an intramolecular charge transfer interaction. The fluorescence quantum yield depends strongly on the properties of the solvents, which was attributed to positive and negative solvatokinetic effects. A crystalline solid of DMATP gave an excimer like emission at 570 nm due to the excitation of molecular aggregates. This is expected from the idealized crystal structure of the dye that belongs to the B-type class of Steven's classification. A dye solution ca. 10⁻³ mol dm⁻³ in CHCl₃ gave a good laser emission in the range 480–560 nm with emission maximum at 530 nm upon pumping by nitrogen laser (λ_ex=337.1 nm). The excitation energy transfer from 7-dimethylamino-4-methyl coumarine (DMC) to DMATP has been also studied in CHCl₃ and the values of energy transfer rate constant and critical transfer distance indicate a Főrster-type mechanism. The photoreactivity and net photochemical quantum yield of DMATP in chloromethane solvents are also determined. We applied semiempirical MO calculation using AMI and ZINO/S calculation to understand the geometric and electronic structure of DMATP molecule in both ground and excited states.     

Fluorescent probe based on proton transfer in 4′-(diethylamino)-3-hydroxyflavone: Spectral and luminescent characteristics upon selective excitation

The spectral characteristics of acetonitrile solutions of 4′-(diethylamino)-3-hydroxyflavone dye with dual fluorescence are studied under selective excitation. This dye is a structural analog of 3-hydroxyflavone and exhibits excited-state proton transfer, which, in contrast to 3-hydroxyflavone, has a thermodynamic rather than a kinetic character. The fluorescence spectra at different excitation photon energies and the excitation spectra of different fluorescence bands are studied. It is found that the intensity ratio of the normal and tautomeric fluorescence bands lying near 507 and 570 nm, respectively, depends on the excitation wavelength, namely, this ratio is 1.45 and almost does not change in the region of the main absorption band (370–420 nm), while, in the region of the second singlet band (near 280 nm), it decreases to 1.15. This can be explained by an increase in the probability of proton transfer with formation of a tautomeric form in the case of excitation into the second band. Another interesting feature is the existence of a latent third emission band peaked at 535 nm, which was found and reliably recorded upon excitation at wavelengths of 470–500 nm. Addition of water quenches this emission, which indicates that it belongs to the anionic form of the dye.     

Measurement of Absolute Fluorescence Quantum Yield of Basic Fuchsin Solution Using a Dual-Beam Thermal Lens Technique

The dual beam thermal lens technique is an effective method for the measurement of fluorescence quantum yield of dye solutions. The concentration-dependent quantum yield of a novel dye of triaminotriphenylmethane family in ethanol is studied using this technique. The absolute fluorescence quantum yield is measured and is observed that the reduction in the quantum yield is due to the non-radiative relaxation of the absorbed energy.     

Spectral properties of molecular charge-transfer probe QMOM

The spectral characteristics of solutions of a dye with dual fluorescence, 1-methyl-2-(4-methoxy)phenyl-3-hydroxy-4(1H)-quinolone, in acetonitrile are studied upon selective excitation. This dye is a structural analogue of 3-hydroxyflavone and also exhibits excited-state proton transfer, which, as well as in the case of 3-hydroxyflavone, has a kinetic nature. The fluorescence spectra are studied upon excitation by photons of various energies, and the excitation spectra are recorded at wavelengths of different fluorescence bands. It is found that the intensity ratio of the emission of the normal and tautomeric forms (at wavelength of 415 and 518 nm, respectively) is almost the same (0.23–0.25) for excitation in the regions of the main and the second absorption bands. At the same time, in the case of excitation between these bands, this ratio decreases to 0.19. The second interesting feature is the existence of a third latent emission band peaked at about 480 nm, which is reliably detected upon excitation at wavelengths in the region of 400–450 nm. This study shows that this emission belongs to the anionic form of the dye. This form is also responsible for a decrease in the intensity ratio of the emission of the two main forms in the case of excitation between the first and second absorption bands.     

磺基水杨酸的荧光光谱与荧光量子产率

报道了磺基水杨酸 (SSA)的荧光光谱和荧光量子产率。在 pH <2时 ,SSA无荧光 ,随pH升高 ,SSA荧光增强 ,在 pH 5～ 10 5之间 ,SSA有稳定的强荧光 ,最大发射波长为 4 0 2nm ,激发波长为 2 12 ,2 38和2 97nm。在 pH >13的强碱性条件下 ,SSA转变为另一种荧光型体 ,最大激发波长 2 6 1nm ,最大发射波长390nm。SSA浓度较高时 ,荧光激发光谱发生变化 ,但发射光谱不变。在近中性条件下 ,SSA稀溶液的荧光强度与浓度之间存在良好的线性关系 ,线性范围为 5～ 2 5 0ng·mL- 1 ,检测下限为 5ng·mL- 1 。以硫酸奎宁为参比 ,测量了SSA在不同波长下的荧光量子产率 ,在最大激发波长 2 97nm处的荧光量子产率为 0 5 4。