Dynamic quenching of the dual fluorescence of molecules

Mathematical relations describing the properties of spontaneous steady-state dual fluorescence under conditions of dynamic quenching of excited states by foreign impurities are derived. It is shown that, in the case of a kinetic character of the reaction, the initial form of the dye and its photoproduct are quenched, the intensity ratio of the fluorescence bands of the initial form and the product linearly increasing with the quencher concentration. Analysis performed is applicable to a wide range of photoreactions accompanied by the dual fluorescence (charge transfer, proton transfer, complexation, etc.). The properties of the fluorescence, absorption, and dual fluorescence excitation for 3-hydroxyflavone in acetonitrile under conditions of dynamic quenching by the TEMPO spin quencher with a concentration below 1.25 × 10^?2 M are studied. 3-Hydroxyflavone is characterized by the excited-state intramolecular proton transfer and by the fluorescence spectrum consisting of two well-spaced bands. The observed dependences of the intensity of both fluorescence bands on the quencher concentration correspond to the theoretical conclusions. The Stern-Volmer constants calculated from the experimental data on the assumption of diffusion quenching of the excited states are 858 and 1141 M^?1 for the normal and tautomeric fluorescence bands, respectively. The experimental results reveal the kinetic character of the excited-state proton transfer in 3-hydroxyflavone in acetonitrile.     

Numerical simulation of intramolecular dynamics during dual fluorescence

The dynamics of transformation of a light pulse by a five-level model molecule whose secondary emission spectrum can contain two fluorescence bands is simulated. The system of equations that determine the time behavior of the matrix elements of the statistical operator of the molecule interacting with the light pulse is numerically solved. From this solution, the time dependences of the populations of the molecular states are determined for different values of the parameters of the irradiation pulse, which is described in terms of the classical theory, and of the parameters that characterize the rates of radiative and nonradiative spontaneous transitions of the molecule. Based on particular examples of the choice of these parameters, it is demonstrated that the mechanism by which dual fluorescence occurs in molecules with intramolecular hydrogen bonds can be efficiently established from the numerically simulated intramolecular dynamics.     

Effect of temperature on the dynamic quenching of the dual fluorescence of molecules

The properties of the dual fluorescence of 3-hydroxyflavone in acetonitrile are studied under conditions of dynamic quenching by the spin quencher TEMPO in the temperature range from 20 to 80°C. 3-Hydroxyflavone is characterized by the intramolecular excited state proton transfer and its spectrum consists of two well-spaced fluorescence bands belonging to the normal and tautomeric forms. The fluorescence was selectively excited at wavelengths of 290, 304, and 340 nm, belonging to different absorption bands of the luminophore. The character and degree of the temperature quenching of the fluorescence depend on the excitation wavelength and are considerably different for normal and tautomeric fluorescence bands. The Stern-Volmer constants for both forms, calculated from the experimental data on the assumption of the diffusion mechanism of quenching of the excited states, increase with temperature. Both in the pure solutions and in the solutions with the quencher, the intensity ratio of the fluorescence of the initial form and the product increases with heating in the entire temperature range from 20 to 80°C for all the above excitation wavelengths. Original Russian Text ? V.I. Tomin, 2008, published in Optika i Spektroskopiya, 2008, Vol. 104, No. 6, pp. 926–933.     

Enhancement and quenching of single-molecule fluorescence

We present an experimental and theoretical study of the fluorescence rate of a single molecule as a function of its distance to a laser-irradiated gold nanoparticle. The local field enhancement leads to an increased excitation rate whereas nonradiative energy transfer to the particle leads to a decrease of the quantum yield (quenching). Because of these competing effects, previous experiments showed either fluorescence enhancement or fluorescence quenching. By varying the distance between molecule and particle we show the first experimental measurement demonstrating the continuous transition from fluorescence enhancement to fluorescence quenching. This transition cannot be explained by treating the particle as a polarizable sphere in the dipole approximation.     

Concentration quenching of fluorescence in solutions

The paper describes the result of the measurements of the intensity and polarisation of fluorescence of dyestuffs in solution and their conductivities at different concentrations. From the nature of the variation of the intensity, polarisation and conductivity with concentration it has been suggested that the quenching of fluorescence of dyes in solution is due to the collision of the second kind.     

Distance-dependent fluorescence quenching ofN-acetyl-l-tryptophanamide by acrylamide

We examined the time-dependent intensity decays ofN-acetyl-l-tryptophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol over a range of temperatures. 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, and quenching of NATA by acrylamide was observed even in vitrified propylene glycol, where translational diffusion cannot occur during the lifetime of the excited state. These frequencydomain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide, and the results are consistent with a rate constant for quenching that depends exponentially on the fluorophore-quencher separation distance. The exponential distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, and the steady-state data aid in determining the interaction distance between NATA and acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of acrylamide quenching of proteins.     

Complexing and quenching of the prodan fluorescence

Experimental and quantum-chemical investigations of the prodan molecule are carried out. Prodan complexes in binary mixtures with nitromethane are investigated. Models of prodan-nitromethane interactions of both NN and NB bond types are constructed. __________ Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 5, pp. 59–64, May, 2006.     

Evaluation of pyrene fluorescence quenching in biological membranes

The redistribution between two excited forms of pyrene accompanying a change in the intramembrane probe concentration yields a set of fluorescence spectra that intersect at a single common (isobestic) point. Infensities of monomer and excimer fluorescence (I_m and I_e, respectively) are related by a linear dependence: I_e+kI_m=I_s. The coefficient k depends on the membrane type and temperature. Fluorescence intensity at the isobestic point (I_i) along with the proportional quantity I_s reflect the total steady-state level of excited pyrene forms and do not depend on the excimerization degree (I_e/I_m ratio), and can be used when estimating the quenching degree of pyrene fluorescence in biological membranes. Institute of Photobiology, Academy of Sciences of Belarus, 27, F. Skorina St., Minsk, 220072, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 64, No. 2, pp. 204–208, March–April, 1997.     

Oxygen quenching of toluene fluorescence at elevated temperatures

Gas-phase oxygen quenching of toluene laser-induced fluorescence (LIF) is studied between 300 and 650 K in a nitrogen/oxygen bath gas of 1-bar total pressure with oxygen partial pressures up to 400 mbar. With increasing vibrational excitation of the laser-excited toluene, intramolecular decay becomes faster, resulting in a decreasing relative strength of collisional quenching by oxygen. Additionally, Stern–Volmer plots are found to be non-linear for temperatures above 500 K in the case of 266-nm excitation and at all temperatures for 248-nm excitation. This is attributed to the onset of internal conversion from specific vibrational levels. A photophysical model is developed that describes the experimental data and predicts toluene LIF signal strengths for higher oxygen partial pressures. One important result for practical application is that oxygen quenching is not the dominant de-excitation process for engine-related temperature and pressure conditions, and thus application of the popular fuel–air ratio LIF (FARLIF) concept leads to erroneous signal interpretation.This revised version was published online in August 2005 with a corrected cover date.     

Oxygen fluorescence quenching studies with single tryptophan-containing proteins

The work of Lakowicz and Weber [Biochemistry 12, 4161 (1973)] demonstrated that molecular oxygen is a powerful quencher of tryptophan fluorescence in proteins. Here we report studies of the oxygen quenching of several proteins that have a single, internal tryptophan residue. Among these are apoazurin (Pseudomonas aeruginosa), asparaginase (Escherichia coli), ribonuclease T₁ (Aspergillus oryzae), and cod parvalbumin. Both fluorescence intensity and phase lifetime quenching data are reported. By comparison of these data we find that there is a significant degree of apparent static quenching in these proteins. The dynamic quenching rate constants,k _q, that we find are low compared to those for tryptophan residues in other proteins. For example, for apoazurin we find an apparentk _q of 0.59×10⁹ M ^–1 s^–1 at 25°C. This value is the lowest that has been reported for the oxygen quenching of tryptophan fluorescence.     

Dynamic quenching of the multiband fluorescence of 3-hydroxyflavone

The properties of emission, absorption, and dual fluorescence excitation of 3-hydroxyflavone in acetonitrile are studied under the conditions of dynamic quenching by potassium iodide with concentrations up to 4 × 10^?2 M. The normal and tautomeric forms undergo quenching, which is more efficient for the tautomeric form. An interesting circumstance is that the absorption in the S ₀ → S ₁ and S ₀ → S ₂ singlet bands of the solution increases with increasing quencher concentration in the whole region of concentrations used, the steepest rise being recorded in the concentration region from 0 to 5 × 10^?3 M. The intensities and quantum yields of the two fluorescence bands show rather complicated nonlinear dependences on the quencher concentration. The long-wavelength fluorescence band, which belongs to the tautomeric form of 3-hydroxyflavone, is quenched considerably stronger. The experimental results reveal the kinetic character of the excited-state proton transfer in molecules of 3-hydroxyflavone in acetonitrile.     

Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation

We observed the steady-state and time-resolved emission of tetraphenylbutadiene (TPB) whea excited by simultaneous absorption of two photons (514 to 610 nm). The intensity initially increased quadratically with laser power, as expected for a two-photon process. At higher laser powers the intensity increases in TPB were subquadratic. The intensity and anisotropy decay times of TPB were unchanged under the locally intense illumination. Importantly, the time zero anisotropy of TPB was decreased under conditions where the intensity was subquadratic. Furthermore, the subquadratic dependence on incident power was not observed for two-photon excitation of 2,5-diphenyloxazole (PPO), for which the incident wavelength does not overlap with the emission spectrum. These results are consistent with stimulated emission (light quenching) of TPB at high laser intensities. The phenomenon of light quenching may be important for other fluorophores used in biochemical research, particularly for the high local intensities used for two-photon excitation.     

On the mechanism of rhodamine 6G fluorescence quenching

The fluorescence of the widely used TPF dye rhodamine 6G is quenched by photons of the ruby as well as the Nd-glass laser. Taking into consideration the polarisation of the ground- and excited-state absorptions of rhodamine 6G a simple model of quenching results. The main process is an S₁ absorption with subsequent leave of the ordinary rhodamine 6G singlet system. For Nd-laser photons the cross section of the concerned transition following from our quenching experiments is σS₁→S₂ = 1.2×10^-16 cm².     

Phenol and anisol fluorescence quenching in aqueous micellar solutions

Phenol and anisol fluorescence quenching in aqueous micellar solutions is investigated on addition of H ₂ O ₂. It is established that the efficiency of interaction of the phenol molecule in water with hydrogen peroxide is higher than of anisol. In the phenol-H ₂ O ₂ system, the buildup of phenol fluorescence occurs in the presence of an anionic-reagent detergent. A decrease in the efficiency of phenol fluorescence quenching by hydrogen peroxide in aqueous solutions in the presence of CTAB and triton X-100 micelles is observed. The micellar media increase the efficiency of interaction of anisol with a quencher in comparison with phenol. __________ Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 4, pp. 73–79, April, 2006.     

Diffusion-influenced fluorescence quenching dynamics in one to three dimensions

The fluorescence decay curves obtained from diffusion-influenced quenching in various spatial dimensions are discussed. The two-dimensional quenching has, because of intractable fitting functions, previously been dealt with only in the completely diffusion-controlled case (corresponding to the Smoluchowski boundary condition). In this paper, an approximation for the two-dimensional (2D)-quenching behavior with the Collins-Kimball boundary condition is presented. The nonlinear least-squares method has been used to analyze simulated decay data. The consequences the choice of an incorrect model has on the final results as well as the possibility to discriminate between different dimensionalities are investigated. Also, some inherent properties of the fitting functions are studied.     

Dynamic fluorescence quenching and the highest excited states of molecules

The dynamic fluorescence quenching in organic molecules, or quenching of the second kind according to Vavilov’s classification, is an efficient method of investigating excited states in solutions and is widely used in various fields. The effect of quenching on the intensity of the fluorescence from the first and higher singlet states of organic molecules is studied. The results may serve as a basis for determining the nature of the short-wavelength luminescence and can be used to distinguish the S _n fluorescence from the comparably intense luminescence of impurities, which is a very important problem when investigating such emissions. A method for obtaining dynamic quenching by specially chosen quenchers is proposed. The method is based on an experimentally found strong increase in the constants of bimolecular collisions of luminophore and quencher molecules when the luminophore is excited through the highest singlet states.