Photobleaching

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 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

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