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Photobleaching.ogg
Photobleaching: The movie shows photobleaching of a fluorosphere. The movie is accelerated, the whole process happened during 4 minutes.

Photobleaching is the photochemical destruction of a fluorophore. 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.

Photobleaching may also be exploited to study the motion and/or diffusion of molecules, for example via the FRAP or FLIP techniques.

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, 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.

Lifetime

Depending on the material, dyes can produce different photon numbers and therefore have different lifetimes (at e.g. 105 photons/s):

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

External links

  • 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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