Fluorescence Quenching

The natural fluorescence intensity of a fluorophore can be decreased by a wide variety of processes. Such decreases in fluorescence intensity upon which the system returns to its initial ground state are referred to as fluo-rescence I0"9&'(9.. Quenching can occur by different mechanisms, but very often electron transfer reactions are involved. Other typical quench-ing processes are energy transfer, paramagnetic quenchquench-ing by oxygen, or concentration quenching. Quenching can be intramolecular or intermo-lecular. In the case of intermolecular (bimolecular) quenching, the quenching can be dynamic or static, depending on whether the quenching partner of the excited molecule, a species called the I0"9&'"*, has to diffuse or not prior to causing deactivation of the excited state of the

fluorophore. Both static and dynamic quenchings require the partners to be at reacting distance, sometimes at molecular contact. After photoexci-tation, if the distance is short enough, the fluorophore returns to the ground state without emission of a photon. Quenching occurs in principle without any permanent change in the molecules. The need for relatively short distances between the quenching partners has lead to numerous applications of fluorescence quenching both as a tool and as a source of information about the investigated systems (see for example [57-59]).

a) Dynamic Quenching

When quenching results from collisional encounter between the fluoro-phore and the quencher, it is termed dynamic, or sometimes collisional or diffusive. Because of its dependence on collisional contacts between the excited molecule and the quencher, this process is usually kinetically of first order in the quencher concentration [Q]. The ratio of the fluores-cence intensities at a given wavelength in the absence and presence of the quencher, A0 and Aq respectively, or of the quantum yields, >0 and >q, is described by the Stern-Volmer equation (for a derivation of this equation, see [16]): excited-state lifetime in the absence of the quencher, and ;Q is called the Stern-Volmer quenching constant. According to equation (1.60), a plot of

0/ q

A A versus [Q] should give a straight line with an intercept of one and a slope equal to ;Q.

5diff represents the diffusion-controlled bimolecular rate constant and therefore the maximal rate at which diffusional bimolecular processes can take place in the condensed phase (see section 1.4.4). As a first

approxi-mation, this constant depends only on the solvent viscosity A (in cP) and

For a typical organic solvent with a viscosity of 0.5 cP, 5diff is on the order of 10 s^{10 !1&}M^!1.

The bimolecular quenching rate constant is directly related to the col-lisional frequency by the quenching efficiency !q:

q q diff

5 "! 5 . (1.63)

An efficiency of 0.5 would result in a bimolecular quenching rate constant half the diffusion-controlled value. Since 5diff can be evaluated only with moderate precision, the observed value of the bimolecular quenching rate constant can be used to qualitatively judge the efficiency of the quenching.

If the quenching results in an electron transfer reaction, the quenching efficiency usually depends on the oxidation and reduction potentials of the two partners.

b) Static Quenching

In some cases, quenching reflects formation of a non-fluorescent ground-state complex between the fluorophore and the quencher. If the forma-tion and the dissociaforma-tion of the complex occur slowly compared with the excited-state lifetime, the fluorescence decay dynamics may show two distinct components if the time-resolution in the experiment is good enough: one very short-lived fluorescence component from molecules that are in complexes at the time of excitation and a longer-lived compo-nent from free molecules. In a typical single-photon counting experiment (see section 6.1.1) with 200 ps time resolution, the short component may not be detected.

The dependence of the fluorescence intensity on the quencher con-centration is easily derived by considering the association constant for complex formation ;A:

A

[FQ]

[F][Q]

; " . (1.64)

In this equation, [FQ] is the complex concentration, [F] the concentration of the uncomplexed fluorophore, and [Q] the uncomplexed quencher concentration. The total fluorophore concentration &0 is given by the mass action law:

0 [F] [FQ]

& " 0 . (1.65)

Within the approximation that the complexed species is instantaneously quenched and thus non-fluorescent, the fraction of the fluorescence that remains, A_q/A₀, is a direct measure of the total fluorophores that are not complexed and fluorophore concentrations can be replaced by fluores-cence intensities. By combining these relationships and rearranging, one gets dy-namic quenching can often be distinguished by varying the viscosity of the solvent as dynamic quenching usually declines with increasing viscosity because it is controlled by the diffusion rate. The most definitive method to distinguish between static and dynamic quenching is however a meas-urement of fluorescence lifetimes. Static quenching removes a fraction of the fluorophores from observation, thus reducing the total fluorescence intensity. Still, the uncomplexed molecules are essentially unperturbed, implying that their fluorescence lifetime with or without quencher be the same. Therefore, for purely static quenching, 6 6 "0/ q 1; in contrast, for purely dynamic quenching, 6 6 "0/ q A0/Aq. One additional method which might enable to discriminate between static and dynamic quenching is an inspection of the absorption spectra of the fluorophore in the presence of the quencher. Whereas no change in the absorption spectrum is expected

for dynamic quenching as it affects only the excited state, ground-state complex formation often results in the appearance of a new band or of a perturbation in the absorption spectrum.

A mixture of dynamic and static quenching can result in a Stern-Volmer plot with upward curvature as the quencher concentration is raised. The fractional fluorescence remaining is then given by the product of the fraction not complexed and the fraction not quenched through collisions. One gets a quadratic expression for the ratio of the fluores-cence yields in the presence and in the absence of the quencher:

# $ # $

Another way that a molecule (the =%9%*, D) can return to the ground state after excitation is to transfer its excitation energy to another molecule (the

?&&"/$%*, A) which is thereby raised to a higher energy state:

* *

D 0A^NNOD A0 (1.69)

The excitation may in principle be electronic, vibrational, rotational, or translational. The discussion in this work will be restricted to "-"&$*%9(&

"9"*.+ $*?9)!"*, also called "7&($?$(%9 "9"*.+ $*?9)!"* (EET). Energy transfer plays a particularly important role in photosynthetic systems, as found in plants and some bacteria. Sunlight gets absorbed by arrays of pigment-protein complexes which form antenna and light-harvesting sys-tems and subsequently transfer the energy to reaction centres, where it is trapped in electron-transfer reactions [60].

Two main energy transfer mechanisms exist. In the first one, the

*?=(?$(8" energy transfer, also known as trivial mechanism, the excitation energy is transferred by radiative deactivation of a donor molecule and