Secondary emission influenced fluorescence decay of a homogeneous fluorophore solution.

An analytical expression is found allowing the calculation of the secondary emission influenced fluorescence decay of a homogeneous fluorophore solution. Before starting the calculation one has to know the shape of the primary fluorescence decay of the fluorophore and the value of the parameter κ denoting the ratio of the secondary to primary steady-state fluorescence intensities. The method elaborated by Budó and Ketskeméty is recommended for evaluation of the parameter κ. The main importance of the obtained expression is that it can be used to recover parameters characterizing the fluorescence decay in the absence of secondary emission. The cases of monoexponential, biexponential and multiexponential primary fluorescence decays are discussed in detail.

Site-to-site distance distribution in flexible molecules: theoretical evaluation of the donor and/or acceptor fluorescence decay function.

We present the theoretical expression describing dependence of the fluorescence intensity decays on the distance distribution P(r)between energy donors and acceptors in flexible bichromophoric molecules. The expression allows for multiexponential fluorescence decay of the donor- and acceptor-only molecules and takes into account the possibility of incomplete labeling of the molecules by acceptors. It is assumed that the donors and acceptors are static in space and do not move relative to each other during the excited-state lifetime. The potential application of the obtained expression is evaluation of the parameters of the function P(r).

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.

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.

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.

Fluorescence energy transfer in one dimension: frequency-domain fluorescence study of DNA-fluorophore complexes.

Fluorescence resonance energy transfer among linear DNA bound fluorophores was carried out to study the process in one dimension. The donor fluorescence intensity decays in the case of energy transfer in one dimension are stretched exponential and show exp[-(t/tau)1/6] time dependence, which results in an initial more rapid decay and subsequent slower decay at long times when compared to those in higher dimensions. DNA-bound 4',6'-diamidino-2-phenyl indole (DAPI), acridine orange (AO), and ethidium bromide (EB) were used as donors. The acceptors were in the case of DAPI AO and EB; in the case of AO nile blue (NB), methylene blue (MB), and crystal violet (CV); and NB, MB, and oxazine 750 in the case of EB. As expected, the donor intensity decays became highly heterogeneous upon energy transfer and were characterized by the simultaneous presence of both highly and marginally quenched donors. The intensity decays for all three donors in the presence of various acceptors are satisfactorily described by the Förster model of energy transfer in one dimension. The intensity decays also allow for clear rejection of a two- or three-dimensional model. The experimentally recovered critical Förster distances (R0) ranged between 37 A in the case of DAPI and EB to 70 A in the case of AO and CV donor-acceptor pairs. These recovered R0 values compare reasonably with those calculated from spectral properties if we use values of 1.25 for k2, and 1.5 for the refractive index of DNA. The k2 value will be even higher, between 1.5 and 2.0, if the consensus DNA refractive index of 1.75 is used. These k2 values strongly suggest that the dipoles of the acceptor chromophores when bound to DNA are not randomly oriented but are aligned preferentially in plane.

End-to-end diffusion coefficients and distance distributions from fluorescence energy transfer measurements: Enhanced resolution by using multiple donors with different lifetimes.

We describe a method to improve the resolution of donor-to-acceptor distance distributions in molecules which are flexing on the timescale of the fluorescence lifetime. We measured the timedependent donor decays of two donor (D)-acceptor (A) pairs, where the donor lifetimes were substantially different. The donors were an indole residue (5.7 ns) and a naphthalene residue (24.4 ns). The same dansyl acceptor was used for both D-A pairs. The donor decays are complex due to both a distribution of D-A distances and D-A diffusion. Using the donor decay data for each D-A pair alone, it is difficult to resolve both the distance distribution and the D-to-A diffusion coefficient. However, these values are unambiguously recovered from global analysis of the data from both D-A pairs. The increased resolution from the global analysis is apparently the result of the complementary information content of the data for each D-A pair. The shorter-lived indole donor provides more information on the time-zero distance distribution because there is less time for D-A diffusion, and the longer-lived naphthyl donor is quenched to a greater extent than indole due to the longer time for diffusion-enhanced energy transfer. Simulations were also used to demonstrate the increased resolution of global analysis with different lifetime donors to obtain distance distribution parameters in the presence of D-A diffusion.

Light quenching of fluorescence: a new method to control the excited state lifetime and orientation of fluorophores.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high-repetition rate lasers, a phenomenon we call "light quenching." In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.

Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays.

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high repetition rate lasers, a phenomenon we call "light quenching." We now describe the theory of light quenching and some of its effects on the steady-state and time-resolved intensity and anisotropy decays of fluorophores. Light quenching can decrease or increase the steady-state or time-zero anisotropy. Remarkably, the light quenching can break the usual z axis symmetry of the excited-state population, and the emission polarization can range from -1 to +1 under selected conditions. The measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The effects of light quenching are different for a single pulse, which results in both excitation and quenching, as compared with a time-delayed quenching pulse. Time-delayed light quenching pulses can result in step-like changes in the time-dependent intensity or anisotropy and are predicted to cause oscillations in the frequency-domain intensity and anisotropy decays. The increasing availability of pulsed laser sources offers the opportunity for a new class of two-pulse or multiple-pulse experiments where the sample is prepared by an excitation pulse, the excited state population is modified by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.

Distance-dependent fluorescence quenching of tryptophan by acrylamide.

We used GHz frequency-domain fluorometry to investigate the time-dependent intensity decays of N-acetyl-L-trytophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol at 20 degrees C. 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. At low temperature in vitrified propylene glycol (-60%), where translational diffusion cannot occur during the lifetime of the excited state, quenching of NATA by acrylamide was observed. The Smoluchowski and RBC quenching models do not predict any quenching in the absence of translational diffusion. Hence, these frequency-domain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide. The rate for quenching of NATA by acrylamide appears to depend exponentially on the fluorophore-quencher separation distance. Comparison of the time-resolved and steady-state data provides a sensitive method to determine the distance dependence of the fluorophore-quencher interaction. The distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, which is often observed for quenching by acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of quenching data of proteins by acrylamide.

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

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 10(-7) to 10(-5) cm2/s. This possibility was verified by studies of a methylene-chain 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 alpha-helical state but over a limited range of distances.

Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores.

We report steady-state and time-resolved studies of quenching of fluorescence by light i.e. "light quenching." The dyes rhodamine B (RhB) and 4-dicyanomethylene-2-methyl-6(p-dimethamino)-4H-pyrane (DCM) were excited in the anti-Stokes region from 560 to 615 nm. At a high illumination power the intensities of DCM and RhB were sublinear with incident power, an effect we believe is due to stimulated emission, andnot ground-state depopulation. The extent of light quenching was proportional to the amplitude of the emission spectrum at the incident wavelength, as expected for light-stimulated decay from the excited state. Control measurements at a decreased average illumination power, and in solvents of various viscosities, indicated that the effect was not due to undesired photochemical processes. Importantly, the frequency-domain intensity decays remained single exponentials, and the lifetimes were unchanged with light quenching, which suggests that the effect was not due to heating or other photochemical effects. These results are consistent with a quenching process which occurs within the quenching pulse. Importantly, as expected for light quenching with a single pulsed laser beam, the time 0 anisotropies of RhB and DCM were decreased due to orientation-dependent quenching of the excited-state population. In closing we discuss some possible future applications of light quenching to studies of dynamic processes.

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 patient's bedside, and even to the doctor's 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.

Linear dichroism study of metalloporphyrin transition moments in view of radiationless interactions with tryptophan in hemoproteins.

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe(3+)-PPIX (ferric-heme) and Fe2+CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300-380 nm) and Soret (380-450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300-380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300-380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J. 63, 648-653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 degrees from the alpha-gamma meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.

End-to-end diffusion coefficients and distance distributions from fluorescence energy transfer measurements: enhanced resolution by using multiple acceptors with different Förster distances.

We measured distance distributions and end-to-end diffusion coefficients of donor-acceptor pairs linked by a flexible methylene chain using frequency-domain fluorescence energy transfer measurements. The donor was an indole group, and two acceptors with different Förster distances were used. The uncertainties in the recovered parameters describing the end-to-end distance distribution and diffusion coefficient were rather large when each donor-acceptor pair was analyzed separately. It was not possible to resolve distance distributions in the presence of intra-molecular diffusion when the Förster distance was comparable to the mean and half-width of the distribution. Global analysis using two acceptors dramatically improved the resolution. Surprisingly, the Förster distances need not be very different, and a 20% difference between the two R0 values resulted in substantial improvements in resolution. Both the simulations and the experiments suggest the benefit of using global analysis with different Förster distances to obtain reliable distance distribution parameters in the presence of diffusion.

Distance distributions and dynamics of a zinc finger peptide from fluorescence resonance energy transfer measurements.

Time-resolved fluorescence resonance energy transfer (FRET) measurements were used to measure distance distributions and intramolecular dynamics (site-to-site diffusion) of a 28-residue single-domain zinc finger peptide in the absence and presence of zinc ion. Energy transfer was measured between TRP14 and a N-terminal DNS group. As expected, the TRP-to-DNS distance distribution for zinc-bound peptide is shorter and narrower (R av=11.2 Å,hw=2.8 Å) than the metal-free peptide (R av=20.1 Å,hw=14.5 Å). The degree of mutual donor-to-acceptor diffusion (D) was also determined for these distributions. For zinc-bound peptide there is no detectible diffusion (D≤0.2 Å(2)/ns), whereas for metal-free peptide a considerable amount of motion is occurring between the donor and the acceptor (D=12 Å(2)/ns). These results indicate that the zinc-bound peptide folds into a unique, well-defined conformation, whereas the metal-free conformation is flexible and rapidly changing. The absence of detectible mutual site-to-site diffusion between the donor and the acceptor in the metal-bound zinc finger peptide indicates that intramolecular motion is essentially frozen out, on the FRET time scale, as a consequence of zinc coordination.

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.

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.