Quenching (fluorescence)


Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodide ions and acrylamide are common chemical quenchers. The chloride ion is a well known quencher for quinine fluorescence. Quenching poses a problem for non-instant spectroscopic methods, such as laser-induced fluorescence.
Quenching is made use of in optode sensors; for instance the quenching effect of oxygen on certain ruthenium complexes allows the measurement of oxygen saturation in solution. Quenching is the basis for Förster resonance energy transfer assays. Quenching and dequenching upon interaction with a specific molecular biological target is the basis for activatable optical contrast agents for molecular imaging.

Mechanisms

Förster resonance energy transfer

There are a few distinct mechanisms by which energy can be transferred non-radiatively between two dyes, a donor and an acceptor. Förster resonance energy transfer is a dynamic quenching mechanism because energy transfer occurs while the donor is in the excited state. FRET is based on classical dipole-dipole interactions between the transition dipoles of the donor and acceptor and is extremely dependent on the donor-acceptor distance, R, falling off at a rate of 1/R6. FRET also depends on the donor-acceptor spectral overlap and the relative orientation of the donor and acceptor transition dipole moments. FRET can typically occur over distances up to 100 Å.

Dexter electron transfer

is another dynamic quenching mechanism. Dexter electron transfer is a short-range phenomenon that falls off exponentially with distance and depends on spatial overlap of donor and quencher molecular orbitals. In most donor-fluorophore–quencher-acceptor situations, the Förster mechanism is more important than the Dexter mechanism. With both Förster and Dexter energy transfer, the shapes of the absorption and fluorescence spectra of the dyes are unchanged.
Dexter electron transfer can be significant between the dye and the solvent especially when hydrogen bonds are formed between them.

Exciplex

formation is a third dynamic quenching mechanism.

Static quenching

The remaining energy transfer mechanism is static quenching. Static quenching can be a dominant mechanism for some reporter-quencher probes. Unlike dynamic quenching, static quenching occurs when the molecules form a complex in the ground state, i.e. before excitation occurs. The complex has its own unique properties, such as being nonfluorescent and having a unique absorption spectrum. Dye aggregation is often due to hydrophobic effects—the dye molecules stack together to minimize contact with water. Planar aromatic dyes that are matched for association through hydrophobic forces can enhance static quenching. High temperatures and addition of surfactants tend to disrupt ground state complex formation.

Collisional quenching

An important quenching process in atmospheric physics can be seen in the altitudinal variation of auroral emissions. At high altitudes, the red 630.0 nm emission of atomic oxygen dominates, whereas at altitudes in the E-layer the green 557.7 nm emission is more intense. Both practically disappear at altitudes below 100 km. This variation occurs due to the unusually long lifetimes of the excited states of atomic oxygen, with 0.7 seconds for the 557.7 nm and almost two minutes for the 630.0 nm emission. The mean collision-free paths decrease at lower altitudes due to increasing particle densities, which results in the de-excitation of the oxygen atoms due to the higher probability of collisions, preventing the emission of the red and green oxygen lines.