Photobleaching

As photobleaching , photobleaching , fading or chemical quenching refers to the permanent loss of fluorescence of a fluorophore by the irradiation of the fluorophore by excitation light. Photo bleaching is a dynamic process in which the fluorophore molecules are destroyed photochemically by the excitation light and thereby lose their ability to fluoresce.

The average number of excitation and emission cycles that a fluorophore can perform before it is inactivated by photobleaching depends on the intensity and energy of the excitation light, the molecular structure of the fluorophore and the chemical environment in which the fluorophore is located is located. Some fluorophores are bleached fewer photons after emission , while others can go through several thousand or millions of excitation and emission cycles until photobleaching occurs.

Photo bleaching can lead to artifacts in fluorescence spectroscopy and fluorescence microscopy . In the fluorescence microscopic methods FRAP (Fluorescence Recovery after Photobleaching) and FLIP (Fluorescence Loss in Photobleaching), on the other hand, an intentionally generated photobleaching is used for the measurement. By reducing the energy of the excitation beam and the irradiation time of the fluorophore, an attempt can be made to minimize the effect of photobleaching.

Typical numerical values

Number of typical photon absorption and emission cycles and irradiation times (at 10 ⁵ photons / s) of different fluorescent dyes:

• Green fluorescent protein : 10 ⁴ -10 ⁵ ; 0.1-1 s
• typical organic fluorescent dye: 10 ⁵ -10 ⁶ ; 1-10 s
• CdSe / ZnS quantum dot : 10 ⁸ ; > 1000 s

Mechanism of photobleaching

Photobleaching is a series of different photochemical reactions in which light-induced damage or modification of the fluorophore occurs. However, photobleaching of fluorophores is a poorly understood phenomenon.

By absorbing a photon, an electron passes into a more energetic state. The interaction of the excited fluorophore with other molecules can then lead to irreversible covalent changes in the fluorophore. Since the triplet state has a longer life than the singlet state, excited fluorophores that are in the triplet state have a much larger time window available in which chemical reactions with molecules in their environment can occur.

credentials

1. ↑ Loling Song, EJ Hennink, I. Ted Young, Hans J. Tanke: Photobleaching Kinetics of Fluorescein in Quantitative Fluorescence Microscopy , Biophysical Journal, 68 , 2588-2600, (1995).