Time-resolved Fluorescence Decay Data Research Articles

Correction Approach for Delta Function Convolution Model Fitting of Fluorescence Decay Data in the Case of a Monoexponential Reference Fluorophore.

A correction is proposed to the Delta function convolution method (DFCM) for fitting a multiexponential decay model to time-resolved fluorescence decay data using a monoexponential reference fluorophore. A theoretical analysis of the discretised DFCM multiexponential decay function shows the presence an extra exponential decay term with the same lifetime as the reference fluorophore that we denote as the residual reference component. This extra decay component arises as a result of the discretised convolution of one of the two terms in the modified model function required by the DFCM. The effect of the residual reference component becomes more pronounced when the fluorescence lifetime of the reference is longer than all of the individual components of the specimen under inspection and when the temporal sampling interval is not negligible compared to the quantity (τR−1 – τ−1)−1, where τR and τ are the fluorescence lifetimes of the reference and the specimen respectively. It is shown that the unwanted residual reference component results in systematic errors when fitting simulated data and that these errors are not present when the proposed correction is applied. The correction is also verified using real data obtained from experiment.

FRET Based Ratio-Metric Sensing of Hyaluronidase in Synthetic Urine as a Biomarker for Bladder and Prostate Cancer

Elevated hyaluronidase levels are found in the urine of bladder and prostate cancer patients. Therefore, HA-ase is regarded as an important biomarker for the detection of these cancers. In this report, we use a FRET based ratiometric sensing approach to detect the level of HA-ase in synthetic urine. For this, we have used a HA-FRET probe (hyaluronan) labeled with fluorescein as a donor and rhodamine as an acceptor. We monitor the digestion of our HA-FRET probe with different concentrations of HA-ase in synthetic urine via fluorescence emission. The extent to which FRET is released depends on the concentration of HA-ase. Our fluorescence intensity results are also supported with time resolved fluorescence decay data. This assay can be used to develop a non-invasive technique for the detection of bladder and/or prostate cancer progression.

Fast time-correlated single-photon counting fluorescence lifetime acquisition using a 100 MHz semiconductor excitation source

The fast and efficient collection of time-resolved fluorescence decay data is shown using a high repetition rate semiconductor laser excitation source optimally matched to low dead-time counting electronics. The 100 MHz repetition rate allows a measurement range of 10 ns without re-excitation of the sample. Rapid acquisition of time-resolved fluorescence data is essential for microscopy applications, such as fluorescence lifetime imaging. With this in mind we measured a representative dye (lifetime 376 ps) to determine the fastest collection time for a single exponential decay. Other dyes were also used with lifetimes ranging from ∼90 ps to 4 ns.

Kinetically Controlled Photoinduced Electron Transfer Switching in Cu(I)-Responsive Fluorescent Probes

Copper(I)-responsive fluorescent probes based on photoinduced electron transfer (PET) switching consistently display incomplete recovery of emission upon Cu(I) binding compared to the corresponding isolated fluorophores, raising the question of whether Cu(I) might engage in adverse quenching pathways. To address this question, we performed detailed photophysical studies on a series of Cu(I)-responsive fluorescent probes that are based on a 16-membered thiazacrown receptor ([16]aneNS(3)) tethered to 1,3,5-triarylpyrazoline-fluorophores. The fluorescence enhancement upon Cu(I) binding, which is mainly governed by changes in the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 29-fold fluorescence enhancement upon saturation with Cu(I) in methanol and a greater than 500-fold enhancement upon protonation with trifluoroacetic acid. Time-resolved fluorescence decay data for the Cu(I)-saturated probe indicated the presence of three distinct emissive species in methanol. Contrary to the notion that Cu(I) might engage in reductive electron transfer quenching, femtosecond time-resolved pump-probe experiments provided no evidence for formation of a transient Cu(II) species upon photoexcitation. Variable temperature (1)H NMR experiments revealed a dynamic equilibrium between the tetradentate NS(3)-coordinated Cu(I) complex and a ternary complex involving coordination of a solvent molecule, an observation that was further supported by quantum chemical calculations. The combined photophysical, electrochemical, and solution chemistry experiments demonstrate that electron transfer from Cu(I) does not compete with radiative deactivation of the excited fluorophore, and, hence, that the Cu(I)-induced fluorescence switching is kinetically controlled.

Similarity of fluorescence lifetime distributions for single tryptophan proteins in the random coil state

The picosecond time-resolved fluorescence decay data of nine single-tryptophan (trp) proteins and two multi-trp proteins in their native and denatured states were analyzed by the maximum entropy method (MEM). In the denatured state (6 M guanidine hydrochloride) a majority of the single-trp proteins show bimodal (at 25 degrees C) and trimodal (at 85 degrees C) distributions with similar patterns and similar values for average lifetimes. In the native state of the proteins the lifetime distributions were bimodal or trimodal. These results (multimodal distributions) are contradictory to the unimodal Lorentzian distribution of lifetimes reported for some proteins in the native and denatured states. MEM analysis gives a unimodal distribution of lifetimes only when the signal-to-noise ratio is poor in the time-resolved fluorescence decay data. The unimodal distribution model is therefore not realistic for proteins in the native and denatured states. The fluorescence decay components of the bi- or trimodal distribution are associated with the rotamer structures of the indole moiety when the protein is in the random coil state.

Effects of static quenching and light pulse intensity on the time-dependent fluorescence quenching kinetics

By employing the general kinetic theory proposed recently for treating the diffusion-influenced fluorescence quenching, we investigate the nonequilibrium dynamic effect of the complex formation reaction between the ground state fluorophor and quencher molecules on the time-resolved fluorescence decay curve. We find that as the complex formation constant K eq becomes larger the time at which the fluorescence intensity becomes maximum shifts to a shorter time. In contrast, conventional theory predicts that the scaled fluorescence decay curve is invariant under the change in the K eq value. This observation suggests a possibility of determining the magnitude of K eq from the time-resolved fluorescence decay data as well as from the curvature of a Stern—Volmer curve. We also investigate the effects of various kinetic factors such as the intensity and shape of the light pulse, the magnitude of quencher concentration, and the presence of an attractive Coulomb force or a long-range energy transfer mechanism. We find that the conventional theory may fail in some experimental situations where repeated excitation of fluorophor molecules is possible.

Reconvolution analysis in time-resolved fluorescence experiments—an alternative approach: Reference-to-excitation-to-fluorescence reconvolution

A novel method for reconstructing the apparatus response function required for accurate detailed analysis of nanosecond time-resolved fluorescence decay data is proposed and tested with properly constructed simulated data sets for a variety of pertinent cases, using the high-accuracy convolution algorithm previously developed [J. Večeř, A. A. Kowalczyk, and R. E. Dale, Rev. Sci. Instrum. 64, xxx (1993)]. The veracity of recovery of test mono- and multiexponential decay responses by this method, involving the use of two appropriately chosen monoexponential reference decay responses, is shown to be essentially identical to that attainable with the ‘‘true’’ apparatus response function. The method is also demonstrated to exhibit significant advantages in a variety of situations over currently the most widely employed method of overcoming extant problems in the direct determination of an appropriate apparatus function: indirect analysis against a monoexponential reference decay response (δ-function convolution method, F-F deconvolution).

Resolution of heterogeneous fluorescence into component decay-associated excitation spectra. Application to subtilisins

Direct and indirect methods are described to combine steady-state and picosecond time-resolved fluorescence decay data to generate decay-associated excitation spectra. The heterogeneous fluorescence from a fluorophore mixture that models protein fluorescence was resolved into individual component excitation spectra. The two methods were also used to determine the excitation spectra associated with each of the decay time components for the proteins subtilisin Carlsberg and BPN'. On the basis of associated spectra, the decay components of both proteins were assigned to individual (or groups of) emitting species. The two approaches used to generate the decay-associated excitation spectra are compared and their general application to protein fluorescence studies is discussed.

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase.

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.