Steady-state fluorescence intensity

Emission and excitation spectra are recorded using a spectrofluorometer (see Chapter 6). The light source is a lamp emitting a constant photon flow, i.e. a constant amount of photons per unit time, whatever their energy. Let us denote by N0 the constant amount of incident photons entering, during a given time, a unit 

Let us recall that the pseudo-first order rate constant for this process is very large (ka x 1015 s 1) whereas the subsequent steps of de-excitation occur with much 

Under continuous illumination, the concentration [1A ] remains constant, which means that JA is in a steady state. Measurements under these conditions are then called steady-state measurements. 

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 . 

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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... 

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.

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) ... 

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... 

The importance of comparing time-dependent and steady-state fluorescence measurements is well illustrated by the difficulty of resolving purely static from purely dynamic quenching. In either case, the basic relationship between the steady-state fluorescence intensity and quencher concentration is the same. The Stem-Volmer relationship for static quenching due to formation of an intermolecular complex is i... 

In systems where only dynamic quenching occurs, then steady-state fluorescence intensities can be measured instead of lifetimes/101 103-,07) In experiments where comparisons are being made (i.e., for a comparison of different experimental conditions or types of membrane), it is important that the lifetime of the fluorophore (r0) is not affected by the experimental conditions. Fluorescence intensities can be obtained much more rapidly and without specialized instrumentation. Blatt and Sawyer(101) have employed a relationship essentially the same as Eq. (5.20) in this way. They have pointed out that since the quenching mechanism is collisional, the partition coefficient that is derived is a partition coefficient of the quencher into the immediate environment of the fluorophore and is therefore a local Kp. It is therefore possible to investigate the partition coefficient gradient across the lipid bilayer by using a series of probes, such as the anthroylstearates,(108) located at different depths. In their method, Eq. (5.20) has the form... 

If the photons emitted from the excited states to the ground state are distinguishable, such as by having significantly different polarizations or frequencies, then the following normalized second-order correlation functions of the steady-state fluorescence intensity can be written as [57]... 

Whereas the observed decay profile no longer is characterized by a single decay rate, the steady-state fluorescence intensity becomes dependent on both 7obs and fc>bs. The typical Stern-Volmer plot is no longer represented by equation 7a, but rather by equation 7b, where fcobs is defined by equation 6b, fc q is the bimolecular quenching rate constant, fco is the probe s mean excited-state unimolecular decay rate constant, fcobs is the mean observed decay rate constant, 70 is the distribution parameter of the Gaussian for the unimolecular decay, and 7obs is the distribution parameter for the observed unimolecular decay rate. 

The decay time, t, is defined by convention as the time for steady state fluorescent intensity to decay to 0.3679 (1/e) of its original value. 

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... 

Fig. 1. (right) The steady state fluorescence intensity of D1/D2 reaction centres, under aerobic conditions at a chlorophyll concentration of 2 pgmr, as a function of the Qy band absorption maximum. The fluorescence intensity is in arbitrary units. The wavelength shifts of the absorption maximum to the blue were caused either by exposure to light at 277 K (O), to a temperature of 295 K in the dark ( ) or by the addition of 1% triton X-100 to the buffer (In the dark at 277 K) (a). 

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