Fluorophore lifetimes

By measuring 0 and m at several modulation frequencies, a set of simultaneous equations can be generated that allow a determination of the best values for fluorophore lifetime and fractional contributions. The fi values from these calculations are the quantities that are used in Equations 7 and 8 to obtain weighted values ofx and Po. 

B. W. Williams and C. D. Stubbs, Properties influencing fluorophore lifetime distributions in membranes, Biochemistry 27, 7994-7999 (1988). 

The Low power microwaves employed here do not perturb the plasmonic surfaces, do not produce arcing which is conunonly observed for metallic objects in microwave cavities,[56] or even denature or change protein conformation. Low power microwaves provide for effective rapid heating of the assays, producing identical final fluorescence intensities, fluorophore lifetimes, as well as extents of energy transfer (protein conformation) as compared to room temperature incubation. 

The direct characterization of an eT mechanism requires a much more complicated technique time-resolved spectroscopy. The solution containing the system under investigation is irradiated by a laser pulse, and the absorption spectra of the solution are consecutively recorded at chosen and very short time intervals (e.g. every 10 ns). If, in the envisaged two-component system F1 M, an M-to-Fl eT process takes place upon illumination, one should be able to measure the absorption spectra of Fl and M" ", as well as their decay, which allows the determination of the lifetime of the transient species F1 M. It goes without saying that very sophisticated and expensive instrumentation is required to carry out this type of experiment. Moreover, the smaller the fluorophore lifetime and the faster the back-electron transfer process, the more rapid and expensive the data acquisition equipment required. In particular, narrow laser pulses and especially fast data collections are needed for systems such as 1, where a short-living polyaromatic fluorophore (anthracene, r = 5 ns) is linked to the electron donor (or acceptor) group by a rather short carbon chain. 

Here, is the quantum yield, modified by the nanostructure A fand k[ are rate constants as in (1) K, is the additional rate constant induced by the nanostructure and Tnsis the fluorophore lifetime modified by the nanostructures (Fig. 10). 

Fluorophore lifetime is another method to measure the degree of fluorescence quenching. 

When the fluorophore lifetime is equal to or lower than the rotational correlation time of the protein, the extrapolated anisotropy will be lower than the limiting one and the rotational correlation time obtained from the slope of the Perrin plot will correspond to an apparent rotational correlation time segmental motion of the fluorophore (Fig. 5.8). 

Fluorescence polarisation spectroscopy is still very much used to probe the rotational dynamics of single molecules, either on surfaces or in solution [152]. In bioa-nalytical assays the fluorescence emission intensity is measured as a function of rotational speed. When a solution of fluorophores is excited with polarised light, the fluorophores selectively absorb those photons that are parallel to the transition moment of the fluorophore, resulting in photoselective excitation. The fluorophore molecules rotate to varying extents during the fluorophore lifetime. If the fluores-... 

Fluorescence lifetime imaging microscopy (FLIM)-based guantitative fluorescence resonance energy transfer (FRET). Direct detection of biomolecular interactions is possible with FRET measurements, where a donor fluorophore transfers the energy to an acceptor fluorophore in case they are close in space. The combination of this technique together with FLIM, based on the decrease in donor fluorophore lifetime that is induced by FRET, has recently enabled the quantitative assessment of the protein-interacting fractions [12]. 

This relationship provides an alternative method to determination of the concentration of the analyte of interest. Specifically, lifetime or decay time measurements can be used in fluorescence based sensors to determine the analyte concentration. These measurements provide better results than steady-state measurements. Time-domain lifetime measurements are typically performed by exciting the sensing element with a short optical pulse which is much shorter than the average fluorophor lifetime. For a single population of fluorophors, the rate at which the intensity decays over time can be expressed as ... 

Unfortunately, direct measurement of the fluorophor lifetime requires significant electronic instmmentation. To overcome this, the variation in the fluorescence lifetime is measured in the frequency domain [30]. In frequency-domain measurements the fluorophor is excited using a modulated light source whose frequency is proportional to the inverse of the average fluorophor lifetime. The fluorescence emission is therefore at the same frequency of the excitation source but it is phase shifted with respect to the excitation source. The relation between the phase shift and the lifetime of the fluorophor can be readily calculated as ... 

The photon emission rate of a fluorophore is determined by the intensity dependent excitation rate, the lifetime of the fluorophore and its quantum efficiency. The excitation rate and the fluorophore lifetime allow the calculation of the saturation of the fluorophore. The photon emission rate as well as bleaching rate is directly proportional to it. 

Carlsson Kand Liljeborg A 1997 Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation J. Microsc. 185 37-46... 

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

Fluorescence lifetime measurements can increase the analytical specificity when analyzing mixtures (1-4) and can indicate changes in chemical binding of the fluorophores under various environmental conditions (5). 

Rotational diffusion during the lifetime of the excited state of the fluorophores... 

T = the lifetime of the emitting fluorophore, and p = the rotational relaxation time of e molecule after excitation. 

Theory. If two or more fluorophores with different emission lifetimes contribute to the same broad, unresolved emission spectrum, their separate emission spectra often can be resolved by the technique of phase-resolved fluorometry. In this method the excitation light is modulated sinusoidally, usually in the radio-frequency range, and the emission is analyzed with a phase sensitive detector. The emission appears as a sinusoidally modulated signal, shifted in phase from the excitation modulation and partially demodulated by an amount dependent on the lifetime of the fluorophore excited state (5, Chapter 4). The detector phase can be adjusted to be exactly out-of-phase with the emission from any one fluorophore, so that the contribution to the total spectrum from that fluorophore is suppressed. For a sample with two fluorophores, suppressing the emission from one fluorophore leaves a spectrum caused only by the other, which then can be directly recorded. With more than two flurophores the problem is more complicated but a number of techniques for deconvoluting the complex emission curve have been developed making use of several modulation frequencies and measurement phase angles (79). 

For single exponential fluorescence decay, as is expected for a sample containing just one fluorophore, either the phase shift or the demodulation can be used to calculate the fluorescence lifetime t. When the excitation light is modulated at an angular frequency (o = 2itv, the phase angle f, by which the emission modulation is shifted from the excitation modulation, is related to the fluorescence lifetime by ... 

Consider a sample with two fluorophores, A and B, whose lifetimes (lA and XB) are each independent of emission wavelength and are different fram one another. By setting 4>D = 4>A 90°, the contribution from A is nulled out and the scanned emission spectrum represents only the contribution from B. Similarly, the spectrum for A is obtained by setting 4>D = 4>B 90°. In practice, finding the correct value of for this... 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

The commercially available dicyanomethylene squaraine dye Seta-670-mono-NHS showed extremely low blinking effects and good photostability when used in single-molecule studies of multiple-fluorophore labeled antibodies [113]. Seta-670-mono-NHS and Seta-635-NH-mono-NHS were covalently labeled to antibodies and used in a surface-enhanced immunoassay [114]. From the fluorescence intensity and lifetime changes determined for a surface that had been coated with silver nanoparticles, both labeled compounds exhibited a 15- to 20-fold... 

As seen from (1) and (2), intermolecular processes may reduce essentially the lifetime and the fluorescence quantum yield. Hence, controlling the changes of these characteristics, we can monitor their occurrence and determine some characteristics of intermolecular reactions. Such processes can involve other particles, when they interact directly with the fluorophore (bimolecular reactions) or participate (as energy acceptors) in deactivation of S) state, owing to nonradiative or radiative energy transfer. Table 1 gives the main known intermolecular reactions and interactions, which can be divided into four groups ... 

Exciplexes are complexes of the excited fluorophore molecule (which can be electron donor or acceptor) with the solvent molecule. Like many bimolecular processes, the formation of excimers and exciplexes are diffusion controlled processes. The fluorescence of these complexes is detected at relatively high concentrations of excited species, so a sufficient number of contacts should occur during the excited state lifetime and, hence, the characteristics of the dual emission depend strongly on the temperature and viscosity of solvents. A well-known example of exciplex is an excited state complex of anthracene and /V,/V-diethylaniline resulting from the transfer of an electron from an amine molecule to an excited anthracene. Molecules of anthracene in toluene fluoresce at 400 nm with contour having vibronic structure. An addition to the same solution of diethylaniline reveals quenching of anthracene accompanied by appearance of a broad, structureless fluorescence band of the exciplex near 500 nm (Fig. 2 )... 

In (8), the solvent-independent constants kr, kQnr, and Ax can be combined into a common dye-dependent constant C, which leads directly to (5). The radiative decay rate xr can be determined when rotational reorientation is almost completely inhibited, that is, by embedding the molecular rotor molecules in a glass-like polymer and performing time-resolved spectroscopy measurements at 77 K. In one study [33], the radiative decay rate was found to be kr = 2.78 x 108 s-1, which leads to the natural lifetime t0 = 3.6 ns. Two related studies where similar fluorophores were examined yielded values of t0 = 3.3 ns [25] and t0 = 3.6 ns [29]. It is likely that values between 3 and 4 ns for t0 are typical for molecular rotors. 

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