Photobleaching and Bleaching Effects
Bleaching (or photobleaching) is the progressive fading of the fluorescence emission intensity of the sample during microscopic imaging.
Any fluorescence microscopy technique in which the sample is imaged over time or over multiple depths can be affected by sample bleaching. Continued or repeated excitation of the fluorescent molecules (fluorophores) causes them to emit less light resulting in fading of the image. In particular, images from Widefield 3D, Widefield time series, Spinning Disc time series, Confocal time series and STED time series are affected. Bleaching in z-stacks and time series images can be succesfully corrected with the Huygens Bleaching Corrector tool.
Bleaching or Quenching?
Both terms refer to the loss of fluorescence intensity of a dye or fluorescent protein. However, while quenching processes are reversible, (photo)bleaching causes permanent damage the structure of the fluorophore. Quenching is caused by interaction of fluorophores in the excited state with molecules from The performance of a fluorescence microscope depends in large part on the chemical components that function as emission dyes inside the (biological) object under study.Causes of bleaching
Bleaching is caused by either prevention of exictation of the fluorophore, or deactivation of the fluorophore without the emission of a photon. Bleaching is highly dependend on the photostability of the fluorophore, and is determined by the properties of the fluorophore, and the environment (solvent) in which it is embedded. Photostability (or fluorophore lifetime), describes how well the fluorophore can sustain emission of photons during repeated stimulation by excitation light before it becomes unusable. Here we describe some of the mechanisms behind bleaching.Jablonski diagram showing the fluorophore transitions from ground state (S0) to the excited states (S1 and S2). Bleaching occurs when the fluorophore undergoes intersystem crossing from the excited state towards the triplet (T) state. Here, the fluorophore does not return to the ground state by emitting a photon, but by undergoing phosphorescence.
Solvent interactions
Interactions of the fluorophore with the embedding environment is the main cause of bleaching. This type of bleaching can be divided into two groups: dynamic and stable bleaching. Dynamic bleaching refers to the deactivation of the excited state by collision of the excited fluorophore with other molecules in the solvent. Stable bleachingPhoto-oxidation
The main cause of photobleaching seems to be the reaction of excited fluorophores with oxygen molecules dissolved in the sample. When a fluorophore is excited, by photon absorption, it transitions from the ground state (S0) to an excited state (S1 or S2), after which it can return to the ground state under emission of a photon (fluorescence). However, it is possible for a dye molecule to cross over to an alternative excited state, called the triplet (T) state, which lives longer and is more reactive than the conventional excited state. Due to the long lifetime, this makes it possible for the fluorophore to react with oxygen. Reaction with oxygen furhter increases the life time of the triplet state, in which no photons are emitted, thus suppressing the emission intensity substantially. Additionally, aside from causing a longer 'dark state', reactions with oxygen can cause photo toxicity by generating reactive oxygen species. Reactive oxygen species are harmfull for the cell and the fluorophores, causing degradation of the (fluorescent) proteins within the cell.
Organic reaction
Similar to photo-oxidation, excited fluorophores can also react with organic molecules from the environment. When the fluorophores cross over to the more reactive and long-lived triplet 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 cannot fluoresce.
Multi-photon events
Lastly, unrelated to solvent reactions, 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 either 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.
Reduction
A 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 Correction
Bleaching 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.