Two photon-induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers

We measured the fluorescence intensity and anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled membranes resulting from simultaneous two-photon excitation of fluorescence. Comparison of these two-photon data with the more usual one-photon measurements revealed that DPH displayed identical intensity decays, anisotropy decays, and order parameters for one- and two-photon excitation. While the anisotropy data are numerically distinct, they can be compared by use of the factor 10/7, which accounts for the two-photon versus one-photon photoselection. The increased time 0 anisotropy of DPH can result in increased resolution of complex anisotropy decays. Global analysis of the one- and two-photon data reveals consistency with a single apparent angle between the absorption and the emission oscillators. The global anisotropy analysis also suggests that, except for the photoselection factor, the anisotropy decays are the same for one-and two-photon excitation. This ideal behavior of DPH as a two-photon absorber, and its high two-photon cross section, makes DPH a potential probe for confocal two-photon microscopy and other systems where it is advantageous to use long-wavelength (680- to 760-nm) excitation.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Effects of light quenching on the emission spectra and intensity decays of fluorophore mixtures

We examined a series of fluorophore mixtures to determine the wavelength selectivity of light quenching and the effects of light quenching on the emission spectra and intensity decays. Light quenching can be accomplished using a single excitation pulse train and quenching wavelength (one-beam) or with longer-wavelength quenching pulses time-delayed relative to the excitation pulses (two-beam). Both one-beam and two-beam light quenching were found to alter the intensity decays of the mixtures. The frequency-domain intensity decay data were analyzed to reveal the fractional intensity of each fluorophore in the mixture and the effects of light quenching on the fractional contribution of each fluorophore to the total intensity. Fluorophores were selected to provide a range of decay times and emission wavelengths. The extent of quenching in the mixtures was dependent on which fluorophore had the higher radiative decay rate and emission intensity at the quenching wavelength. A general theory is presented which describes the intensity decays in terms of the extent of quenching of each fluorophore and the time delay between excitation and quenching pulses. The effects of light quenching on the fractional intensities of each fluorophore in the mixture, recovered from the intensity decay analysis, were found to be in quantitative agreement with that predicted from steady-state measurements of light quenching and from the spectral properties of the fluorophores. The data on light quenching of mixtures demonstrate that light quenching may be used for selective quenching of fluorophores and thus of potential value for studies of multichromophoric systems.     

Collisional quenching and depolarization of the fluorescence of the 2,5-diphenyloxazole and 2-phenylbenzoxazole vapors by oxygen

We have measured the constants of collisional quenching and depolarization of the fluorescence of a number of derivatives of 2,5-diphenyloxazole and 2-phenylbenzoxazole in a gas phase by oxygen. The probabilities of quenching and orientation randomization of a single collision are determined. It is shown that quenching is significantly influenced by the electron-donor properties of substituents.__________Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 72, No. 1, pp. 43–47, January–February, 2005.     

Distance-dependent quenching of Nile Blue fluorescence byN,N-diethylaniline observed by frequency-domain fluorometry

Fluorescence quenching of Nile Blue by amines is thought to be due to electron transfer to the excited dye molecule from the amine electron donor. We used electron transfer quenching of Nile blue byN,N-diethylaniline in propylene glycol as a model system for an interaction which depends exponentially on distance. We investigated the time dependence of the presumed distance-dependent process using gigahertz harmonic-content frequency-domain fluorometry. The frequency-domain data and the steady-state quantum yield were analyzed globally based on either the Smoluchowski-Collins-Kimball radiation boundary condition (RBC) model or the distancedependent quenching (DDQ) model, in which the rate of quenching depends exponentially on the flourophore-quencher distance. We performed a global analysis which included both the frequencydomain time-resolved decays and the steady-state intensities. The latter were found to be particularly sensitive to the model and parameter values. The data cannot be satisfactorily analyzed using the RBC model for quenching. The analysis shows the excellent agreement of the DDQ model with the experimental data, supporting the applicability of the DDQ model to describe the quenching by the electron transfer process, which depends exponentially on the donor-acceptor distance.     

Efficiency of energy transfer by resonance in quenching of protein fluorescence by dyes

Considering the quenching of tryptophan fluorescence of bovine serum albumin by various dyes as an example, it is shown that overlap of radiation and absorption spectra does not necessarily lead to energy transfer by resonance. No correlation is revealed between the limiting quenching and the Forster overlap integral. Quenching can occur even in the absence of overlap. The magnitude of energy transfer is markedly lower than that of quenching owing to competing processes, namely, excitation deactivation by the dye and, probably, by the protein itself which undergoes conformation upon sorption of the dye. Negatively charged and neutral dyes posses, on the average, a higher quenching activity relative to albumin than do positively charged dyes. Institute of the Biophysics of Cells of the Russian Academy of Sciences, Pushchino, 142292, Moscow Region, Russia. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 2, pp. 290–293, March–April, 1998.     

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

Time-resolved fluorescence polarization anisotropy and optical imaging of Cybesin in cancerous and normal prostate tissues

Time-resolved polarization-dependent fluorescence of Cybesin in solution and in cancerous and normal prostate tissues were measured. The polarization preservation property of Cybesin in tissue was observed. The fluorescence intensity emitted from a Cybesin-stained cancerous tissue area was found to be much stronger than that from a Cybesin-stained normal tissue area indicating that cancerous prostate tissue takes-up more Cybesin than normal tissue. The polarization anisotropy of Cybesin contained in cancerous prostate tissue was found to be larger than that of Cybesin in normal prostate tissue indicating that a larger degree of polarization was preserved in the Cybesin-stained cancerous tissue due to structures. A static anisotropy component from the emission of cell-bonded Cybesin molecules in tissue and a time-dependent anisotropy component from the emission of un-bonded Cybesin molecules were determined and discussed. The static anisotropy value of Cybesin in stained cancerous tissue was found to be much larger than that in stained normal tissue. The fluorescence polarization difference imaging technique based on the polarization preservation of Cybesin was used to enhance the image contrast between cancerous and normal prostate tissue areas.     

Analysis of fluorescence quenching of pyronin B and pyronin Y by molecular oxygen in aqueous solution

The fluorescence quenching of pyronin B and pyronin Y molecules by molecular oxygen in aqueous solution was studied by using steady-state and time-resolved fluorescence and UV-Vis absorption spectroscopy techniques. In order to understand the quenching mechanism, fluorescence decays, absorption and fluorescence spectra of the probes were recorded as a function of the oxygen concentration and temperature. The quenching was found to be appreciable and shows positive deviation in the Stern-Volmer representation obtained from the fluorescence intensity ratio. Fluorescence quenching constants (k_q) were calculated from the τ_o/τ vs. [Q] plots having linear correlation and compared with calculated diffusion-controlled rate constants (k_diff) values. Experimental results were in good agreement with the simultaneous dynamic and static quenching model.     

Dynamics ofLens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues

The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate bound toLens culinaris agglutinin and of the Trp residues of the protein was investigated. Red-edge excitation spectra and steady-state anisotropy as a function of temperature indicate that the TNS is bound rigidly. Red-edge excitation spectra, steady-state anisotropy as a function of sucrose and anisotropy decay experiments performed on Trp residues fluorescence prove that the internal fluorophore presents residual motion independent of the global rotation of the protein. Fluorescence anisotropy decay allows to calculate the rotational correlation time (351 ps) of this local motion. Quenching resolved emission anisotropy with iodide gives values equal to 0.257 and 0.112 for the anisotropies of the buried and the surface Trp residues, respectively. This result indicates that the Trp residues present at the surface of the protein have important local motions compared to those embedded in the protein matrix. The results obtained from TNS and Trp residues indicate that the agglutinin has different dynamic domains.     

Fluctuation kinetics of the hopping fluorescence quenching in disordered solid solutions: A theoretical model and experimental evidence

Fluctuation kinetics of the hopping fluorescence quenching in disordered solid solutions is investigated for the first time. Measurements were made in the chelated complexes of rare-earth ions [Y_1−xTb_x(pyca)₃(H₂O)₂]nH₂O used as chromophore. The Tb³⁺ ions and the OH^--ions of unbounded water molecules appear as fluorescent donors and randomly distributed acceptors, respectively, with the dipole-dipole interaction between them. The measured fluctuation kinetics is the kinetics of the Förster type but with larger decay amplitude. It begins almost immediately after the initial static stage of quenching, and lasts for the entire interval of measurements leaving no room at all for the well-known exponential kinetics with constant rate. Proposed theoretical explanation of the phenomenon is based on the solution of the closed many-particle integral equation for the observable kinetics of hopping fluorescence quenching. The methods for determination of the energy transfer microparameters by the measured fluorescence quenching kinetics are discussed.