Fluorescence can be lost by:[20]

1. Collisional quenching involving collisions with other molecules that result in the loss of excitation energy as heat instead of as emitted light. This process is always present to some extent in solution samples; species that are particularly efficient in inducing the process are referred to as collisional quenchers (e.g. iodide ions, molecular oxygen, nitroxide radical).
2. Static quenching. Interaction of the fluorophore with the quencher forms a stable non-fluorescent complex. Since this complex typically has a different absorption spectrum from the fluorophore, presence of an absorption change is diagnostic of this type of quenching (by comparison, collisional quenching is a transient excited state interaction and so does not affect the absorption spectrum). A special case of static quenching is self-quenching, where fluorophore and quencher are the same species. Self-quenching is particularly evident in concentrated solutions of tracer dyes.
3. Resonance energy transfer. Like collisional quenching, this is an excited state interaction but the two participating molecules do not have to collide - it can occur over distances of 100 angstroms or more. One dye (the donor) is excited by absorption of a photon, but instead of emitting a fluorescence photon, the excitation is transferred by electronic coupling to an acceptor molecule. The result of this exchange is an excited acceptor and a ground state donor. In signal terms the result is either fluorescence at longer wavelength (compared to the spectrum of the donor alone) or no fluorescence, depending on whether the acceptor is itself fluorescent or non-fluorescent.
4. Inner filter effect. This is a measurement artifact as opposed to a quenching process. It occurs in samples with very high absorbance (the absorbance can be due to the fluorophore itself or other absorbing components of the sample - it doesn't matter which). Under these conditions, all the incident exciting light is absorbed at the front face of the sample. In a conventional 90 degree detection geometry, the excitation light cannot penetrate deeply enough to the point at which the detection optics are focused. The result is the detected fluorescence decreases as the sample absorbance increases. As implied, the magnitude of inner filter effect depends on the geometrical relationship between the excitation and emission detection paths and on the thickness of the sample. To avoid inner filter effects (and other artifacts), it is generally advisable for the sample absorbance measured at the excitation wavelength to not exceed 0.1.