Bleaching (also photobleaching) is the progressive fading of the emission intensity of the sample under study. This in particular affects images from Widefield 3D, Widefield time series, Spinning Disc time seriese, Confocal time series and STED time series.
The main cause of photobleaching seems to be the reaction of excited fluorescent molecules with oxygen molecules dissolved in the sample. It is possible for a dye molecule to cross over to a secondary excited state which lives longer and is more reactive than the conventional excited state. This makes it possible for it to react with oxygen. When the secondary excited state reacts with oxygen the life time increases even further and thus surpressing the emission intensity subtantially.
A second viable explaination of photobleaching is the reaction of dye molecules with organic molecules from the environment. When the dye molecules cross over to the more reactive and long-lived excited state the molecules can undergo a irreversible chemical reaction with intracellular organic molecules such as proteins and lipids. The result is a new molecule that can not fluoresce.
Photobleaching can also be caused by absorption of one or more photons by a fluorescent molecule in an already excited state. If it is hit by another photon it can either cross to a more reactive state or it can ionize. In case of the reactive state it can lead to one of the previous mentioned processes. In case of ionization the molecule will also be unable to fluoresce. However, for these events to occur the excitation intensity needs to be several orders higher than for one-photon events.
Huygens Professional and Huygens Essential have a tool (Equalize Flux) that corrects for bleaching in some situations. See Bleaching Mode for more details. See also Bleaching Vs Sampling.
Since Huygens 17.04, both Essential and Professional include an advanced Bleaching Corrector tool for more interactive control on bleaching correction in z-stacks and time series.