Steady-state fluorescence spectra

Figure 6a shows the standard fluorescence spectra (steady-state spectra) for an inorganic-organic hybrid BP composed of a Ru complex as the energy donor... 

Fluorescence spectrum Steady-state fluorescence intensity Fluorescence decay Modified in the region of spectral overlap Decreased in the region of spectral overlap Unchanged Unchanged Decreased by the same factor whatever Xem Shortened... 

To illustrate the concept of fluorescence yield, we turn again to the K spectrum. Assume that an element is irradiated with an x-ray line energetic enough to excite the K spectrum. If the irradiation is continued, a steady state will soon be reached in which the rate at which holes are produced in the K shell (i.e., the rate at which atoms in the K state are produced) is just balanced by the combined rates of the various processes causing such holes to disappear. Let n1, n2,. . . , % be the individual rates rii at which the filling of holes leads to the production of the i lines in the K spectrum. The fluorescence yield, for this simple case is... 

The solvation dynamics of the three different micelle solutions, TX, CTAB, and SDS, exhibit time constants of 550, 285, 180 ps, respectively. The time constants show that solvent motion in these solutions is significantly slower than bulk water. The authors attribute the observed time constants to water motion in the Stern layer of the micelles. This conclusion is supported by the steady-state fluorescence spectra of the C480 probe in these solutions. The spectra exhibit a significant blue shift with respect the spectrum of the dye in bulk water. This spectral blue shift is attributed to the probe being solvated in the Stern layer and experiencing an environment with a polarity much lower than that of bulk water. 

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

For excitation of solutes with 0-0 transitions v0o>v (antiStokes spectral region of absorption), the situation is the opposite at the initial instant of time, the spectra are red-shifted as compared to the steady state spectra, Av1 (l)<0. In this case, the return of the spectrum to its normal position during configurational relaxation will lead to a blue shift with time. From the physical point of view, this means that the intermolecular energy excess, which the solvates possess before excitation, is partially converted into emitted energy leading to an increase in the radiation frequency with time. That is why the process may be called the up-relaxation of the fluorescence spectra. 

Finally, the third case corresponds to pumping of solutes with 0-0 transitions, Voo>v. In this case, the fluorescence spectrum, immediately after excitation, must be close to the steady state one and should not vary with time. From the physical point of view, this case corresponds to the situation, where solvates with a local field Rtl (corresponding to the equilibrium configuration in the excited state) are excited. 

In contrast, at room temperature, the reconstructed fluorescence spectra were found to be identical to the steady-state spectrum, which means that solvent relaxation occurs at times much shorter than 1 ns in fluid solution. 

From a practical point of view, it should be emphasized that, if relaxation is not complete within the excited-state lifetime, this can lead to misinterpretation of the shift of the steady-state fluorescence spectrum in terms of polarity. 

Steady-state method 2 comparison between the absorption spectrum and the excitation spectrum (through observation of the acceptor fluorescence) The corrected excitation spectrum is represented by... 

One may consider the relaxation process to proceed in a similar manner to other reactions in electronic excited states (proton transfer, formation of exciplexes), and it may be described as a reaction between two discrete species initial and relaxed.1-7 90 1 In this case two processes proceeding simultaneously should be considered fluorescence emission with the rate constant kF= l/xF, and transition into the relaxed state with the rate constant kR=l/xR (Figure 2.5). The spectrum of the unrelaxed form can be recorded from solid solutions using steady-state methods, but it may be also observed in the presence of the relaxed form if time-resolved spectra are recorded at very short times. The spectrum of the relaxed form can be recorded using steady-state methods in liquid media (where the relaxation is complete) or using time-resolved methods at very long observation times, even as the relaxation proceeds. 

A discussion on steady state fluorescent monitoring necessitates a distinction between spectroscopic and photometric measurements. The former involves a grating-based spectrofluorometer where full spectrum excitation and emission multivariate spectra are acquired. In contrast a filter photometer involves optical elements (e.g., optical Alters) to isolate excitation and emission bands thereby resulting in a univariate output emission response. 

Fig. 23 Normalized absorption spectra of the free BSA protein (1), BSA-dye 50 conjugate (2) and steady state fluorescence emission spectrum of the BSA-dye conjugate (2 )...

The steady state spectra of indole solvated in water show similar behavior as those from indole solvated in ethanol (see Fig. la)). Due to the larger dipole moment of the water molecules a somewhat larger Stokes shift of the fluorescence spectrum is observed. 

From the steady state fluorescence spectrum of indole in water a fluorescence quantum yield of about 0.09 is determined. Since the cation appears in less than 80 fs a branching of the excited state population has to occur immediately after photo excitation. We propose the model shown in Fig. 3a). A fraction of 45 % experiences photoionization, whereas the rest of the population relaxes to a fluorescing state, which can not ionize any more. A charge transfer to solvent state (CITS), that was also introduced by other authors [4,7], is created within 80 fs. The presolvated electrons, also known as wet or hot electrons, form solvated electrons with a time constant of 350 fs. Afterwards the solvated electrons show no recombination within the next 160 ps contrary to solvated electrons in pure water as is shown in Fig. 3b). 

Fluorescence decay dynamics of PYP at blue and red edges as well as the maximum of the steady state spectrum... 

The steady-state spectra obtained for different alcohols are depicted in fig. 1. While the absorption spectra red shift with increasing solvent polarisability (from methanol to octanol), the fluorescence shows a red-shift when going from octanol to methanol. The total Stokes shifts are very large 7.900 100 cm 1 for PSBR/MeOH and 6.870 100 cm 1 for octanol. Another striking observation is the 30 % smaller width of the fluorescence spectrum of methanol (AE = 3.420 cm 1) compared with other alcohols. While the widths of the fluorescence spectra are solvent-dependent, the absorption spectra have a FWHM of -5.100 cm"1, irrespective of the solvent. As we will substantiate in the following, this behavior indicates that the potential energy surface around the fluorescent point is different than near the Franck-Condon zone probed by absorption, as suggested by quantum chemistry calculations [7]. 

The steady state absorption and fluorescence spectra of both dendrimer generations 1 and 2 are depicted in Fig. 2. The former are merely superpositions of the absorption spectra of both chromophores involved. In the fluorescence, however, the peryleneimide part is almost completely quenched compared to the model compound. Instead, the fluorescence at wavelengths longer than 650 nm almost completely resembles the emission spectrum of the terrylene-diimide model compound 3. This feature is a strong indication that within these dendrimers the excitation energy is efficiently transferred from the peryleneimide to the terrylenediimide. 

Increasing the solvent polarity results in a red shift in the -t -amine exciplex fluorescence and a decrease in its lifetime and intensity (113), no fluorescence being detected in solvents more polar than tetrahydrofuran (e = 7.6). The decrease in fluorescence intensity is accompanied by ionic dissociation to yield the t-17 and the R3N" free radical ions (116) and proton transfer leading to product formation (see Section IV-B). The formation and decay of t-17 have been investigated by means of time resolved resonance Raman (TR ) spectroscopy (116). Both the TR spectrum and its excitation spectrum are similar to those obtained under steady state conditions. The initial yield of t-1 is dependent upon the amine structure due to competition between ionic dissociation and other radical ion pair processes (proton transfer, intersystem crossing, and quenching by ground state amine), which are dependent upon amine structure. However, the second order decay of t-1" is independent of amine structure... 

Fig. I The steady-state fluorescence excitation spectrum of (1) PAQ based MIP and (2) its emission spectra in the absence and (3) in the presence of cGMP (adapted from [47])...

Steady-State Fluorescence. The fluorescence characteristics of PRODAN are extremely sensitive to the physicochemical properties of the solvent (38). As benchmarks, the steady-state emission spectra for PRODAN in several liquid solvents are presented in Figure 1. It is evident that the PRODAN emission spectrum red shifts with increasing solvent polarity. This red shift is a result of the dielectric properties of the surrounding solvent and the large excited-state dipole moment (ca. 20 Debye units) of PRODAN (38). It is the sensitivity of the PRODAN fluorescence that will be used here to investigate the local solvent composition in binary supercritical fluids. 

The time-resolved emission spectra were reconstructed from the fluorescence decay kinetics at a series of emission wavelengths, and the steady-state emission spectrum as described in the Theory section (37). Figure 4 shows a typical set of time-resolved emission spectra for PRODAN in a binary supercritical fluid composed of CO2 and 1.57 mol% CH3OH (T = 45 °C P = 81.4 bar). Clearly, the emission spectrum red shifts following excitation indicating that the local solvent environment is becoming more polar during the excited-state lifetime. We attribute this red shift to the reorientation of cosolvent molecules about excited-state PRODAN. 

Fig. 11 Illustration of the excited state relaxation derived from experimental results obtained for poly(dA).poly(dT) by steady-state absorption and fluorescence spectroscopy, fluorescence upconversion and based on the modeling of the Franck-Condon excited states of (dA)io(dT)io. In red (full line) experimental absorption spectrum yellow circles arranged at thirty steps represent the eigenstates, each circle being associated with a different helix conformation and chromophore vibrations.

The fluorescence spectrum is found to be markedly non-Boltz-mann and sharply peaked at the directly excited level throughout the laser pulse. This is due to two effects the competition between electronic quenching and rotational relaxation processes (4) and the short length of the laser pulse. Because the pulse is so short, steady state is not established throughout the upper rotational levels. The peaks of the fluorescence pulses from levels which are not directly excited by the laser lag the laser pulse peaks by one to four nanoseconds, depending on the energy gap between the given level and the directly excited level. 

In good solvents at ambient temperature, the excited state (67 ) will quickly relax to the planar form, so that only the 0-0 emission from Si is detected in steady-state emission. If the same experiment is performed at low temperature and in a viscous solvent, the molecular torsion of 67 in attaining its planar form is hampered by the medium, and planarization is slow on the timescale of the fluorescence lifetime (355 ps). Emission will not only occur from the potential minimum of the lowest excited state, but from virtually all frozen ro-tamers resulting in a broad and blue-shifted spectrum. Only after planarization of 67 is complete narrow emission from the lowest excited-state conformation will reoccur. Consequently, planarization of the excited state rather than energy migration is likely to govern the emission behavior in PPEs such as 12. 

Figure 15-15. Time-resolved dispersed fluorescence (TRDF) spectra of PdG in room-temperature aqueous solution at the excitation wavelength of (a) 280 nm and (b) 300 nm. The time gates are 0-100 ps circles) and 0-6.0 ns (squares) in the fluorescence decay. The steady-state spectrum (solid line) of PdG in aqueous solution is also shown for comparison (not scaled to the ordinate). (Reprinted with permission from Ref. [24].)...

On the reducing side of PS II, the primary acceptor (Qa had been considered the primary acceptor until pheophytin was discovered to precede it) is reduced in less than 400 ps by Pheo . The reduction of Qa is conveniently monitored by the increase of PS II fluorescence from an initial value, Fq, to a maximal level, indicative of the steady-state level of Q /Qa- K reoxidation of Qa is prevented by the specific inhibitor DCMU (or other herbicides having the same effect), the fluorescence yield of PS II increases sharply, because Qa becomes fully reduced. The reduced form is an anion semiquinone (see the review by Cramer and Crofts [36]), and the absorption spectrum of this compound with a maximum at 326 nm serves for its identification [19] and offers an alternative method for kinetic studies of Qa redox reactions (see Ref. 37 for review). 

The simplest steady-state measurements of fluorescence properties such as the fluorescence emission spectrum or the steady-state anisotropy can be carried out in a standard fluo-rometer with excitation from a lamp source and a monochromator. Common lamp sources in commercial fluorometers include xenon lamps for excitation from the UV to the near-IR (250 nm to llOOnm). 

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