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 series, Confocal time series and STED time series. Bleaching in z-stacks and time series images can be succesfully corrected with Huygens Bleaching Corrector tool.
MechanismsThe performance of a fluorescence microscope depends on the chemical components that function as emission dyes inside the sample. There are many mechanisms that affect the amount of light emitted from the fluorescent molecule. These mechanisms not only depend on the kind of fluorescent molecules but also on the environment of the sample. This will result in a lifetime for each fluorescent molecule which determines how many absorption-emission cycles can be undergone before the molecule becomes unusable. Here are some of the popular theories regarding the origin of photobleaching.
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.
ReductionA lot of research has been done on this subject. The kind and magnitude of bleaching depends on the composition of the dye molecule and the environment of the sample. Therefore the first step for bleaching reduction is choosing the most photostable fluorescent molecule. Secondly, one can reduce bleaching by deoxygenating the sample. This can be done by passing N2 through the sample or by using oxygen scavengers such as ascorbic acid.
Bleaching CorrectionBleaching correction is by default activated with specific image data. See Bleaching Mode for more details. This correction can be switched off when using the Deconvolution Wizard in Huygens Professional and Huygens Essential, or the Operation Window in Professional. Frequently, the sampling size is reduced to minimize bleaching issues. This is not always necessary, as is discussed on this page: Bleaching Vs Sampling.
Both Essential and Professional include an advanced Bleaching Corrector tool for more interactive control on bleaching correction in z-stacks and time series.