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 acquired in 3D Widefield or (3D) 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 and they are often incorrectly mixed up. While quenching processes are reversible, (photo)bleaching is a longterm loss of fluorescence. Quenching is caused by weak interactions of fluorophores in the excited state with molecules in the solvent, making the amount of quenching dependent on the proximity of quenching molecules to the fluorophores. Bleaching can also be caused by solvent interaction, however in this case the interactions are permanently changing the structure of the fluorophore, inhibitting fluorescence for much longer times.Causes of bleaching
Bleaching is caused by either prevention of exictation of the fluorophore, or by deactivation of the fluorophore without the emission of a photon. The amount of bleaching is strongly related to the photostability of the fluorophore, which 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 can reside for prolonged periods of time, thus not emitting photons. From the triplet state the excited fluorophore does not return to the ground state via fluororescence, but by undergoing phosphorescence, producing light that is not detected as it does not fall within the ranges of the emission wavelengths.
Photo-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 further increases the life time of the triplet state, in which no photons are emitted, thus suppressing the emission intensity substantially.Moreover, from the triplet state, fluorophores can return to the ground state via a process of phosphorescence, which is the emission of a photon, but not at the same emission wavelength as the fluorescence photons. Fluorophores in the triplet state therefore become dark for an extended period. Photons released via phosphorescence, are not detected by the microscope detector as they fall outside of the emission wavelength filter range.
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 biological sample and the fluorophores, causing degradation of the (fluorescent) proteins within the sample.
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 an 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 like oxidation and organic reaction, 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, making this a less common cause of bleaching.Bleaching reduction
The best way to avoid bleaching is to choose image acquisition settings that preserve sample fluorescence. If possible, lower the excitation laser intensity, choose shorter exposure times or reduce the duration or framerate of a time-series experiment. Additionally, you can reduce the amount of Z-planes in 3D imaging or in general reduce the sampling size of your image in every dimension. However, image restoration is most effective when sampling rates are close to the ideal Nyquist rate. Therefore, we advise to keep a high sampling rate, but lower the laser intensity. In this way, the low signal (low SNR) images can still be restored well using Huygens deconvolution. More considerations can be found here: Bleaching Vs Sampling, or watch the presentation "Expanding the Pyramid of Compromise" that can be found here.If changing the image acquisition settings is not possible or bleaching still occurs, consider using another (more photostable) fluorophore, as the severity of bleaching depends largely on the composition of the dye molecule and the environment of the sample. If possible for the experiment, fluoresecent proteins can be immunostained, which generally increases the signal. Alternatively, 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 in Huygens
Although preventing bleaching is best to preserve your sample and not lose any information, bleaching can luckily be corrected with the Huygens Bleaching Corrector. The Bleaching Corrector is included in both Huygens Essential and Professional, and gives you more interactive control over bleaching correction in z-stacks and time series. Moreover, in the Huygens software, bleaching correction is by default activated with specific image data when doing deconvolution. See Bleaching Mode for more details. This automatic bleaching correction can be switched off when using the Deconvolution Wizard or when using deconvolution templates.References
1. https://link.springer.com/article/10.1134/S10681620170600852. https://pubs.rsc.org/en/content/articlepdf/2019/cp/c8cp05063e
3. ttps://www.sciencedirect.com/science/article/pii/S101113441400267X#b0035
4. https://www.edinst.com/blog/jablonski-diagram-2/
5. https://www.researchgate.net/publication/355485633_Investigations_to_characterize_the_interactions_of_light_radiation_engine_operating_media_and_fluorescence_tracers_for_the_use_of_qualitative_light-induced_fluorescence_in_engine_systems/download
6. https://axispharm.com/what-is-fluorescence-quenching/
Last updated: June 2024