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):
- Green fluorescent protein: 104-105; 0.1-1 s
- Typical organic dye: 105-106; 1-10 s
- CdSe/ZnS Quantum dot: 108; > 1000 minutes
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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