Photobleaching

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In optics, photobleaching (sometimes called fading) is a photochemical change of a dye or fluorophore molecule in such a way that it can not permanently fluoresce. This is due to the cleavage of covalent bonding or a non-specific reaction between fluorophore and the surrounding molecule. Irreversible modification as in covalent bonds is caused by the transition from singlets to triplet states of fluorophores. The number of excitation cycles varies to achieve full bleaching. In microscopy, photobleaching can complicate the observation of fluorescent molecules, as they will eventually be destroyed by the exposure of the light required to stimulate them into fluorescing. This is especially problematic in the time lapse microscope.

However, photobleaching can also be used before applying (especially antibody-linked) fluorescent molecules, in an attempt to extinguish autofluorescence. This can help improve the signal-to-noise ratio.

Photobleaching can also be used to study the motion and/or diffusion of molecules, eg via FRAP, where mobile component movement can be confirmed by observing fluorescence recovery at photobleaching sites, or FLIP techniques, where multiple photobleaching rounds are performed so that the spread of fluorescence loss can be observed in cells.

The loss of activity caused by photobleaching can be controlled by reducing the intensity or span of exposure to light, by increasing the concentration of fluorophores, by reducing the frequency and thus the photon energy of the input light, or by using stronger fluorophores less prone to bleaching (eg Alexa Fluors or DyLight Fluors). For a reasonable approach, certain molecules will be destroyed after constant exposure (emission intensity X emission time X number of cycles) because, in a constant environment, each emission absorption cycle has the same probability of causing photobleaching.

Video Photobleaching

Lifetime

Depending on their specific chemistry, the molecule can be engined after absorbing only a few photons, while more powerful molecules can undergo multiple absorption/emission cycles before destruction:

• Green fluorescent protein: 10 ⁴ -10 ⁵ foton; 0.1-1.0 seconds lifetime.
• Typical organic colors: 10 ⁵ -10 ⁶ photons; 1-10 seconds of a lifetime.
• CdSe/ZnS quantum dot: 10 ⁸ foton; & gt; 1,000 seconds of a lifetime.

The use of the term "lifetime" is not to be confused with "lifespan" as measured by lifetime imaging of fluorescence.

Maps Photobleaching

See also

• Ozone depletion

src: os.bio-protocol.org

References

src: www.pnas.org

External links

• Introduction to Optical Microscopy an article about photobleaching
• Viegas MS; Martins TC; Seco F; do Carmo A (2007). "Better and cost-effective methodologies for reducing autofluorescence in direct immunofluorescence studies on embedded paraffin-fixed paraffin networks". Eur J Histochem . 51 (1): 59-66. PMID 17548270.

Source of the article : Wikipedia