Steady state fluorescence

Steady-state fluorescence is the simplest and the most common type of fluorescence spectroscopy. Measurements are performed with continuous illumination and detection that is, the sample is illuminated with a continuous beam of light (usually of a specific wavelength) and the emission spectrum (intensity versus wavelength) is recorded. 

The detection limit of fluorescence techniques is strongly dependent on the efflciency of the fluorescence equipment. The type of information provided by these techniques depends on the fluorescence mode used - either steady-state (steady-state fluorescence) or time-resolved (time-resolved fluorescence) - and also whether the excitation is performed with natural or polarized light (steady-state or time-resolved fluorescence anisotropy) [3], 

The fluorescence spectra obtained corresponds to the sum of the individual contributions of the emitting fluorophores present in the sample, capable of absorbing energy at the selected /lexc- The information elicited through spectral analysis can be based on (a) variation in fluorescence intensity, (b) shifts in maximum emission and (c) emission band broadening. 

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Nonradiative reiaxation and quenching processes wiii aiso affect the quantum yieid of fluorescence, ( )p = /cj /(/cj + Rsiative measurements of fluorescence quantum yieid at different quencher concentrations are easiiy made in steady state measurements absoiute measurements (to detemrine /cpjj ) are most easiiy obtained by comparisons of steady state fluorescence intensity with a fluorescence standard. The usefuiness of this situation for transient studies... 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

Steady state fluorescence quantum yield may be calculated using the equation of Parker and Rees [131] as described by Maiti et al. [132] ... 

The remarkable enhancement of steady state fluorescence emission intensity and quantitative data on fluorescence quanfum yield was sequence-dependenf, being maximum wifh AT-rich DNA and alternating AT polymer (Fig. 6b). 

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.

Alexa488 bound to IFABP monitored by steady-state fluorescence was fitted to a two-state reversible unfolding model. This modified protein is slightly less stable (midpoint of 4.5 M compared to 4.7 M for wild-type IFABP). 

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

Jang DJ, Kelley DF (1985) Time-resolved and steady-state fluorescence studies of the excited-state intramolecular proton transfer and relaxation of 2-hydroxy-4, 5-naphthotro-pone. J Phys Chem 89 209-211... 

Steady-state fluorescence spectra, fluorescence quantum yield (F) and lifetimes (tf) of DTT 15 and DTP 23a were estimated as shown in Table 8. F for DTT is higher than DTP. F for DTP is very small and it was difficult to estimate an accurate fluorescence lifetime by the photon counting method due to weak fluorescence. It is noted that the for DTP depends largely on the solvent and is 7.7 x 10-5 in acetonitrile. This low F value has been attributed to an addition reaction with the solvent. 

It is very difficult to excite the donor without also exciting some of the acceptor population. This is because the absorption spectra of dyes extend significantly into the blue side of their absorption maxima, so the absorption spectra of the donor and acceptor usually overlap. The donor fluorescence can typically be observed without acceptor fluorescence interference therefore, when measuring FRET efficiency by observing the donor fluorescence, this overlap is not important. However, when observing the acceptor fluorescence the overlap of the donor and acceptor absorption must be taken into account. The total steady-state fluorescence of the acceptor, assuming that [A] = [D (i.e., a equal donor and acceptor concentrations, and 100% labeling) is... 

Domingo, B., Sabariegos, R., Picazo, F. and Llopis, J. (2007). Imaging FRET standards by steady-state fluorescence and lifetime methods. Microsc. Res. Tech. 70, 1010-21. 

Steady state fluorescence spectra were recorded using a spectrofluorimeter (Fluorolog TCSPC Horiba Jobin Yvon) equipped with a sample holder for solids. 

Hirsch, R.E., Zukin, R.S., and Nagel, R.L. (1986) Steady-state fluorescence emission from the fluorescent probe 5-iodoacetamido-fluorescein, bound to hemoglobin. Biochem. Biophys. Res. Comm. 138, 4889. 

Photosensitization of diaryliodonium salts by anthracene occurs by a photoredox reaction in which an electron is transferred from an excited singlet or triplet state of the anthracene to the diaryliodonium initiator.13"15,17 The lifetimes of the anthracene singlet and triplet states are on the order of nanoseconds and microseconds respectively, and the bimolecular electron transfer reactions between the anthracene and the initiator are limited by the rate of diffusion of reactants, which in turn depends upon the system viscosity. In this contribution, we have studied the effects of viscosity on the rate of the photosensitization reaction of diaryliodonium salts by anthracene. Using steady-state fluorescence spectroscopy, we have characterized the photosensitization rate in propanol/glycerol solutions of varying viscosities. The results were analyzed using numerical solutions of the photophysical kinetic equations in conjunction with the mathematical relationships provided by the Smoluchowski16 theory for the rate constants of the diffusion-controlled bimolecular reactions. 

All steady state fluorescence experiments were conducted with the sample placed in a thermostated cell with temperature maintained at 30°C. The concentrations of anthracene and initiator used were 0.000505 and 0.00608 moles per liter, respectively. The relative quantities of solvents (n-propanol and glycerol) were adjusted from 0 to 100% to achieve solutions of different viscosities, while maintaining the same molar concentration of the reactive solutes. 

The fluorescence decrease in Figure 1 can be attributed to the consumption of the anthracene photosensitizer during the photosensitization reaction. The photosensitization proceeds by an electron transfer reaction from the anthracene to the initiator, resulting in loss of aromaticity of the of the central ring.17 Therefore, the photosensitization reaction leads to a disruption in the n electron structure of the anthracene, and the resulting molecule does not absorb at 364 nm (nor fluoresce in the 420 - 440 nm region). Hence, the steady-state fluorescence measurements allow the anthracene concentration to be monitored in situ while the photosensitization reaction takes place. 

A series of steady-state fluorescence experiments were performed in mixtures of propanol and glycerol to investigate the effect of viscosity on the effective second order photosensitization rate constant, k2. Figure 3 illustrates that the effective rate constant decreases as the viscosity of the system is increased. For example, as the reaction solvent is changed from pure propanol to pure glycerol, the viscosity of the system rises by three orders of magnitude, while the effective reaction rate coefficient, k2, decreases by approximately one order of magnitude. 

Solution of this coupled set of differential equations allows the concentrations of each of the anthracene electronic states to be determined as a function of time. In a previous publication, Nelson et al 1 used this approach to investigate the relative importance of electron transfer from the singlet and triplet states of anthracene. In this contribution, we will use these simulations to predict profiles of the anthracene ground state as a function of time so that the simulation results may be compared with the steady-state fluorescence results presented above. 

Comparison of the Experimental and Simulation Results. The preceding discussion has shown that both the experimental anthracene fluorescence profiles and the simulated anthracene concentration profiles decrease in a manner which closely follows an exponential decay. Therefore, the most convenient way to compare the simulation results to the experimental data is to define an effective overall photosensitization rate constant, kx or k2, as described above. Adoption of this lumped-parameter effective kinetic constant allows us to conveniently and efficiently compare the experimental data to the simulation results by contrasting the rate constant obtained from the steady-state fluorescence decay with the value obtained from the simulated decrease in the anthracene concentration. 

Methods. Absorption spectra were recorded using an Hitachi model 150-20 spectrophotometer/data processor system. Uncorrected steady-state fluorescence emission spectra were recorded using a Perkin-Elmer MPF-44A spectrofluorimeter. These spectra were collected and stored using a dedicated microcomputer and then transferred to a VAX 11/780 computer for analysis. Fluorescence spectra were corrected subsequently for the response characteristics of the detector (21). Values of the fluorescence quantum yield, <) , were determined relative to either quinine bisulfate in IN H2S04 )>f =... 

The amount of fluorescence photons emitted per unit time and per unit volume, i.e. the steady-state fluorescence intensity, is then given by... 

This expression shows that the steady-state fluorescence intensity per absorbed photon iF/alo is the fluorescence quantum yield8 . 

We have so far considered all emitted photons, whatever their energy. We now focus our attention on the energy distribution of the emitted photons. With this in mind, it is convenient to express the steady-state fluorescence intensity per absorbed photon as a function of the wavelength of the emitted photons, denoted by Fa( f) (in m 1 or nm ) and satisfying the relationship... 

In practice, the steady-state fluorescence intensity If(7f) measured at wavelength AE (selected by a monochromator with a certain wavelength bandpass AAF) is proportional to F (/.f) and to the number of photons absorbed at the excitation wavelength AE (selected by a monochromator). It is convenient to replace this number of photons by the absorbed intensity 1a( e), defined as the difference between the intensity of the incident light 10( e) and the intensity of the transmitted light Jt( e) ... 

B4.2.4). The dotted lines correspond to Eq. (B4.2.5). Insert Steady-state fluorescence spectra of corresponding solutions normalized to the monomer emission (reproduced with permission from Atik et al., 1979b ). 

The steady-state fluorescence intensities are obtained by integration of Eqs (4.43) and (4.44). The ratio of the fluorescence intensities of the excimer and monomer bands, Ie/Im (Figure 4.6), is often used to characterize the efficiency of excimer formation. This ratio is given by... 

In the presence of photoinduced proton transfer, the steady-state fluorescence intensities are given by Eqs (4.55) and (4.56). In the absence of deprotoration (i.e. in a very acidic solution such that k i [H30+] 1/tq), when the experimental conditions (concentrations, excitation and observation wavelengths, sensitivity of the instrument) are kept strictly identical, the fluorescence intensities is (Iah )o = C o- Rewriting Eqs (4.55) as fAH = C , the following ratio is obtained... 

Steady-state fluorescence Decreased in the region of Decreased by the same factor... 

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