Photobleaching is the photochemical destruction of a dye or 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.
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.
The time course of photobleaching, be it exponential or non-exponential, can be approximated by applying, first, the finite Legendre transform to the fluorescence time course, f(t), and secondly, the inverse finite Legendre transform to the truncated Legendre spectrum of f(t).
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 s
This use of the term "lifetime" is not to be confused with the "lifetime" measured by fluorescence lifetime imaging.
- Guobin Bao and Detlev Schild, Fast and accurate fitting and filtering of noisy exponentials in legendre space, 2014. PLoS ONE, 9(3), e90500
- 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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