In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it permanently is unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing. This is especially problematic in time-lapse microscopy.
However, photobleaching may also be used prior to applying the (primarily antibody-linked) fluorescent molecules, in an attempt to quench autofluorescence. This can help to improve signal-to-noise ratio.
Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, by reducing the frequency and thus the photon energy of the input light, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors). To a reasonable approximation, a given molecule will be destroyed after a constant exposure (intensity of emission X emission time X number of cycles) because, in a constant environment, each absorption-emission cycle has an equal probability of causing photobleaching.
Depending on their specific chemistry, molecules can photobleach after absorbing just a few photons, while more robust molecules can undergo many absorption/emission cycles before destruction:
- Green fluorescent protein: 104-105 photons; 0.1-1 second lifetime.
- Typical organic dye: 105-106 photons; 1-10 second lifetime.
- CdSe/ZnS Quantum dot: 108 photons; > 1000 seconds lifetime.
This use of the term "lifetime" is not to be confused with the "lifetime" measured by fluorescence lifetime imaging.
- Introduction to Optical Microscopy an article about photobleaching
- Viegas MS; Martins TC; Seco F; do Carmo A (2007). "An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues". Eur J Histochem 51 (1): 59–66. PMID 17548270.
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