Photobleaching

As photobleaching, photobleaching, or fading chemical quenching is defined as the permanent loss of the fluorescence of a fluorophore due to the irradiation of the fluorophore by excitation light. Photobleaching is a dynamic process, wherein the fluorophore molecules are photochemically damaged by the excitation light, and thereby lose their ability to fluoresce.

The average number of excitation and emission cycles can perform a fluorophore, before being inactivated by photobleaching depends on the intensity and the energy of the excitation light from, as well as on the molecular structure of the fluorophore and the chemical environment in which the fluorophore is located. So some fluorophores are bleached fewer photons after emission, while other thousands or millions of excitation and emission cycles can go through until it comes to photobleaching.

Photobleaching in fluorescence spectroscopy can lead to artifacts. By reducing the energy of the excitation beam and the irradiation time of the fluorophor can be made to minimize the effect of photobleaching. In the methods of FRAP ( fluorescence recovery after photobleaching ) and FLIP (Fluorescence Loss in Photobleaching ) photobleaching intentionally produced is used for measurement.

Typical numerical values

Number of typical photon absorption and emission cycles and exposure times ( at 105 photons / s ) of different fluorescent dyes:

• Green fluorescent protein: 104-105; 0.1-1 s
• Typical organic fluorescent dye: 105-106; 1-10 s
• CdSe / ZnS quantum dot: 108; > 1000 s

Mechanism of photobleaching

Wherein the photo- bleaching there is a variety of photochemical reactions in which there is a light-induced damage or modification of the fluorophore. However, the photobleaching of fluorophores is a hitherto poorly understood phenomenon.

Due to the absorption of a photon, an electron is transferred to a more energetic state. Through interaction of the excited fluorophore with other molecules may then come to irreversible covalent modifications of the fluorophore. Since the triplet state is long-lived than the singlet state, have excited fluorophores that are located in the triplet state, a much larger window of time in which there may be chemical reactions with molecules in their environment.

Credentials

• Spectroscopy