Józef Kuśba

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.

Time-resolved and steady-state fluorescence quenching of N-acetyl-L-tryptophanamide by acrylamide and iodide.

We examined the time-resolved and steady-state fluorescence quenching of N-acetyl-L-tryptophanamide (NATA) by acrylamide and iodide, over a range of viscosities in propylene glycol. The quenching of NATA by acrylamide and iodide results in heterogeneity of the intensity decay which increases with the quencher concentration. We attribute the complex decays of NATA to transient effects in diffusion and the nature of the fluorophore-quencher interaction. These data were compared using the phenomenological radiation boundary condition (RBC) and distance-dependent quenching (DDQ) models for collisional quenching. We used global analysis of the time-resolved frequency-domain and steady-state data to select between the models. Consideration of both the frequency-domain and steady state data demonstrate that the quenching rate depends exponentially on the fluorophore-quencher distance, indicating the validity of the DDQ model. The rate constants for acrylamide and iodide quenching, at the constant distance of 5 A, were found to be near 10(13) s-1 and 10(9) s-1, respectively. These rates reflect electron transfer and exchange interactions as the probable quenching mechanisms, respectively.

Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry.

Abstract We report measurements of the site‐to‐site diffusion coefficients in proteins and model compounds, which were measured using time‐dependent energy transfer and frequency‐domain fluorometry. The possibility of measuring these diffusion coefficients were shown from simulations, which demonstrate that donor (D)‐to‐acceptor (A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of donor‐to‐acceptor distances. For decay times typical of tryptophan fluorescence, the simulations indicate that D‐A diffusion coefficients can be measured ranging from −7 to −5 cm2/s. This possibility was verified by studies of a methylenechain linked D‐A pair in solutions of varying viscosity. The D‐A diffusion was also measured for two labeled peptides and two proteins, melittin and troponin I. In most cases we used global analysis of data sets obtained with varying amounts of collisional quenchers to vary the donor decay time. Unfolding of troponin I results in more rapid D‐A diffusion, whereas for melittin more rapid diffusion was observed in the α‐helical state but over a limited range of distances.

Donor Fluorescence Decay Analysis for Energy Transfer in Double-Helical DNA with Various Acceptor Concentrations

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.

Emerging biomedical and advanced applications of time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods, are resulting in the rapid migration of timeresolved fluorescence to the clinical chemistry lab, the patients bedside, and even to the doctors office and home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. Future horizons of state-of-the-art spectroscopy are also described. Two photon-induced fluorescence provides an increased information content to time-resolved data. Two photoninduced fluorescence, combined with fluorescence microscopy and time-resolved imaging, promises to provide detailed three-dimensional chemical imaging of cells. Additionally, it has recently been demonstrated that the pulses from modern picosecond lasers can be used to quench and/or modify the excited-state population by stimulated emission since the stimulated photons are directed along the quenching beam and are not observed. The phenomenon of light quenching should allow a new class of multipulse time-resolved fluorescence experiments, in which the excited-state population is modified by additional pulses to provide highly oriented systems.

Distance-dependent fluorescence quenching observed by frequency-domain fluorometry

Abstract We describe frequency-domain measurements of the time-dependent intensity decays of p -bis[2-(5-phenyloxazolyl)] benzene (POPOP) when collisionally quenched by carbon tetrabromide (CBr 4 ). The frequency-domain data reveal the existence of a quenching rate constant which depends exponentially on the fluorophore—quencher separation. A distance-dependent quenching rate explains the decadesold observation of upward curvature in Stern—Volmer plots

Diffusion-modulated energy transfer and quenching: analysis by numerical integration of diffusion equation in laplace space.

Publisher Summary This chapter discusses the diffusion-modulated energy transfer and quenching. Trapping of electronic excitation by acceptors or quenchers has been intensively studied theoretically and experimentally. Both fluorescence resonance energy transfer (FRET) and excitation energy quenching (EEQ), commonly referred to as “collisional or dynamic quenching,” have found numerous applications in studies of biological macromolecules. Fluorescence resonance energy transfer is the result of a distance-dependent through-space interaction, which occurs between an excited donor and unexcited acceptor molecule. The most important interaction of that type is the Coulombic dipole–dipole interaction. FRET is a powerful tool to monitor the spatial distribution of molecules in solutions or in systems characterized by restricted geometries. However, because of the complexity of these systems and the need for high-resolution time-resolved data, FRET measurements are more widely used to measure discrete distances in macromolecules.

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry☆

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.