Thermal Intensity Quenching

De-excitation of a fluorophore occurs via different competitive mechanisms described in the Jablonski diagram. The global rate constant, which is the inverse of the fluorescence lifetime, can be considered equal to the sum of the different rates of the competitive mechanisms. Thus, one can write  

Working at a constant temperature allows the different parameters of the system to be controlled, and one can quantify the mechanisms intervening in the depopulation of the fluorophore excited state. However, what will happen when we measure the fluorescence lifetime or intensity at different temperatures Free in solution, a fluorophore can be temperature-dependent or not. In the first case, the temperature variation affects both 

With a temperature increase, kr decreases. fcjsc is temperature-independent, and so the temperature will not affect kr. The constant /c or thermal constant increases with temperature. k is called the solvent constant, since the latter is considered to be responsible for the temperature dependence of the fluorescence lifetime. k allows fluorophore activation energy to be determined by the classical Arrhenius theory  

Since the fluorescence lifetime can be obtained experimentally and kr calculated, the value of E can be obtained by plotting ln(l/r — kr) as a function of 1/T. One should note that since fcr is at least 10 times less than 1/r, it is no longer taken into account in Equation (10.27). 

The slope of fluorescence lifetime variation with temperature of FMN in the absence of AMP differs from that observed in the presence of AMP, thus indicating that an interaction exists between FMN and AMP. The quantum yield of FMN free in solution varies identically to fluorescence lifetime. However, in the presence of AMP, the quantum yield increases, while the lifetime decreases, thus indicating that a static complex (nonfluorescent) is formed between FMN and AMP. 

Working at constant temperature allows controlling the different parameters of the system and one can detail and quantify the mechanisms intervening in the depopulation of the excited state of the fluorophore. 

E is the Arrhenius activation energy expressed in Real / mole, R is the molar gas constant and T the temperature in Kelvin degrees. Plotting Ln k, as a function of 1 / T yields the value of E. 

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At the breaking temperature, the decrease in the fluorescence intensity is much slower, i.e., we are here studying the thermal intensity quenching of the more compact protein matrix. One can notice that the second slope observed with TNS (- 1% per °C) is almost equal to the slope obtained when experiments were performed with Trp residues (-0.935% per °C). 

Thermal intensity quenching can be performed also to study the dynamics of the carbohydrate residues of a i-acid glycoprotein. This protein contains 40% carbohydrate by weight and has up to 16 sialic acid residues (10-14% by weight). The fluorescent probe calcofiuor white binds to the carbohydrate residues of a i-acid glycoprotein and... 

Thermal Fluorescence intensity quenching of TNS bound tightly to LCA shows two slopes equal to -2.5% per °C and -1.6% per °C. The breaking temperature is equal to 20°C. One can notice that there is no big difference between the two slopes (Fig. 

Figure 4.43. Thermal fluorescence intensity quenching of free FITC (a), of FITC bound to LCA (b), in presence of LCA-LTF complex (c) and in presence of LCA-STF complex (d). Source Albani, J. R. 1998. Biochim. Biophys. Acta. 1425, 405-410.

In the fluorescence intensity quenching (thermal and with iodide), it is the fluorescein environment consisting of amino acids (thermal quenching) and of amino acids and solvent dipoles that is relaxing around the excited fluorescein. In the fluorescence anisotropy experiments, on the other hand, the displacement of the emission dipole moment of the fluorescein is monitored. In the first approach, it is the environment that is either fluid or rigid. In the second approach, the restricted reorientational motion of the fluorophore is followed. 

In conclusion, the different thermal histories imposed to PTEB have a minor effect on the /3 and y relaxations, while the a. transition is greatly dependent on the annealing of the samples, being considerably more intense and narrower for the specimen freshly quenched from the melt, which exhibits only a liquid crystalline order. The increase of the storage modulus produced by the aging process confirms the dynamic mechanical results obtained for PDEB [24], a polyester of the same series, as well as the micro-hardness increase [22] (a direct consequence of the modulus rise) with the aging time. 

The ratio of the intensities of the two delayed emission bands should thus be completely independent of 4>t and of all triplet quenching processes. Since ke represents a thermally activated process,... 

The delayed fluorescence produced by triplet-triplet quenching is to be sharply differentiated from that observed with eosin or proflavine hydrochloride. The latter type has the same lifetime as the triplet and its intensity is proportional to the first power of the rate of light absorption. It is produced by thermal activation of molecules from the triplet level to the excited singlet level and can occur with any substance for which... 

Much earlier, Friedman (IS) proposed that thermal conductivity was an important parameter in the correlation of quenching distance data. Weir and Morrison (43) spectroscopically traversed flat flames physically separated from a porous disk and found that the relative intensities of C2, CH, and OH were decreased as the amount of heat lost from the flame was increased. A large number of investigators have been concerned with... 

These factors are consistent with the 10- to 20-fold decline observed in polyselenide electrolyte in those experiments there also appeared to be little potential dependence of the results (9). Similar thermal quenching data has been reported for dry CdS Te samples irradiated with UV light (10,12,13), electron beams (11), and a particles (19). The temperature dependence of the decline in emission intensity has been linked to the ionization energy of the Te-bound hole, 0.2 eV (10,11,12,13,19). 

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