1 - What do we know about photobleaching?
HOTOBLEACHING or fading is defined as the loss of absorption or emission intensity16. It occurs when a fluorophore permanently loses its ability to emit fluorescence due to photon-induced chemical damage resulting in covalent modifications17. Photobleaching of fluorescent probes may complicate their observation, since they will ultimately be destroyed by the light source necessary to excite them.
All fluorescent probes are subject to such a phenomenon and will emit a certain number of photons before definitely ceasing to do so. It is reported for example that a single fluorescein molecule emits about 3x105 photons18 and GFP-like molecules emit about 105 photons before being destroyed by photobleaching (Kubitscheck et al. 2000). Two-photon irradiation seems to be even more destructive to fluorescent proteins since higher order interactions occur and irreversibly photobleach proteins with higher quantum yields (Patterson & Piston 2000).
Photobleaching, thus, seems to be an unavoidable event that may happen, at any moment of the illumination of a fluorescent protein.
Some techniques use photobleaching at their advantage (White & Stelzer 1999) such as Fluorescence Loss In Photobleaching (FLIP) or Fluorescence Recovery After Photobleaching (FRAP), that allow the determination of molecular diffusion rates. In super-resolution microscopy techniques such as PALM (Betzig et al. 2006), the role of photobleaching is also crucial since molecules are imaged until they are bleached, which terminates a cycle allows imaging of other molecules during the following cycle. However, in most cases, photobleaching represents a strong limitation in microscopy that one tries to avoid or at least reduce as much as possible.
16Glossary of terms used in photochemistry (IUPAC Recommendations 1996) on page 2231
17 http://www.stratagene.com/lit/faq/faq.aspx?fqid=323
18 http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html
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2 - What are the factors influencing photobleaching?
a - The involvement of the triplet state and singlet oxygen
Although photobleaching is still poorly understood, the triplet state T1 is generally recognized, due to its long lifetime, to be the most likely starting point for deleterious reactions leading to chromophore destruction (Donnert et al. 2007). Once in the triplet state, chromophores may either relax and re-populate the ground state, or the long living triplet state can allow reactions between excited chromophores and other molecules to occur, producing irreversible covalent modifications19. Other photobleaching pathways have been suggested to occur from the singlet excited state S1 or even from higher orders of excited states such as S2, that can be reached by illuminations with UV light (Bell et al. 2003; Kong et al. 2007).
One of the causes often proposed for the occurrence of photobleaching is when a fluorophore reacts with singlet oxygen. Singlet oxygen (1O2) is, at the opposite of most molecules, the unstable form of molecular oxygen, which, in its stable form, is actually in a triplet state (3O2). 3O2 can be converted to 1O2 by reacting with another molecule that is in its triplet state, following the general scheme:3O2⟶ O1 2
𝑇1↷ 𝑆0
It has been recently shown that the GFP chromophore produces 1O2 when excited (Jimenez-Banzo et al. 2008), by monitoring the specific phosphorescence band of 1O2 at 1275 nm (Nonell & Braslavsky 2000). However, at the exception of the engineered protein KillerRed which has been reported to produce major quantities of 1O2 (Bulina et al. 2006a;
Bulina et al. 2006b; Remington 2006), the usual production of singlet oxygen by fluorescent proteins seems to be low. Interestingly, whereas this protein produces high quantities of 1O2, its bleaching rate is not particularly important, probably because there is a kind of tunnel allowing the efficient outflow of the reactive oxygen species (ROS) from the -barrel. The technique of chromophore-assisted light inactivation (CALI) has been developed to permit, via the excitation of chromophores such as the one of KillerRed, the major production of ROS that
inactivate the function of neighboring molecules (Bulina et al. 2006b; Jacobson et al. 2008).
However, this technique, and more generally the production of 1O2 did not obviously reveal being very efficient to photobleach GFP-like proteins and it seems that there is no clear evidence of the effect of molecular oxygen or quenchers on GFP bleaching (Swaminathan et al.
1997), most likely because of a shielding effect by the -barrel. Even if it is one of the possible pathways implicated, the chromophore's photoxidation by 1O2, thus, is probably not the main pathway leading to photobleaching.
Photobleaching, reveals to be a very complex phenomenon and has certainly more than a single origin. Many pathways probably coexist, such as the interaction between the chromophore and singlet oxygen, but also deleterious reactions occurring at the triplet state or at the singlet excited states, that lead to the photodestruction of fluorophores. We propose, in this work, to study one of these pathways.
b - The implication of a radical state?
A number of studies focusing on organic dyes have pointed to the possibility of radical formation from T1 as an important pathway for photobleaching (Zondervan et al. 2003;
Hoogenboom et al. 2005) and we can logically think that such a radical formation may also be involved in the photobleaching process of FPs. The strong acidity of the excited chromophore may for example promote electron transfer from the conserved Glu222 (GFP numbering), leading to decarboxylation of this residue via a bi-radical intermediate state [Photo-Kolbe reaction (Kraeutler et al. 1978; Sato 1983; Habibi & Farhadi 1998; Yanga et al. 2008)] and resulting in fluorescence activation (van Thor et al. 2002; Bell et al. 2003; Lukyanov et al.
2005) or bleaching (McAnaney et al. 2005). Contrary to small molecule dyes, the details of the underlying molecular mechanisms have remained largely unexplored for FPs. In particular, the structural distortions of the chromophore induced by radical formation have not yet been elucidated and the development of FPs with superior photostability has mainly followed empirical approaches to date (Ai et al. 2006; Shaner et al. 2008).