Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/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.

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

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, 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.

LifetimeEdit

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.