Photobleaching ( photobleaching, light bleaching ) - photochemical destruction of the fluorophore . In microscopy, photobleaching can complicate the observation of fluorescent molecules , since the latter are destroyed over time when irradiated with light, causing fluorescence.
Photobleaching can be used prior to the use of fluorophores associated with antibodies to quench autofluorescence, which allows to lower the background signal level.
Discoloration by light can be used to study the movement or diffusion of molecules, for example, using ( eng. FRAP , Fluorescence recovery after photobleaching ) or ( eng. FLIP , Fluorescence loss in photobleaching ).
Reducing the effect of photobleaching can be controlled by reducing the intensity or period of exposure to light, increasing the concentration of the fluorophore, reducing the frequency (and photon energy) of the absorbed light, or using more resistant to destruction fluorophores, such as Alexa or DyLight Fluor.
Duration
Depending on the material, dyes can form different numbers of protons and therefore have a different half-life:
- Green fluorescent protein ( eng. GFP ): 10 4 −10 5 ; 0.1-1 s
- Conventional organic dye: 10 5 −10 6 ; 1-10 s
- CdSe / ZnS: 10 8 ; > 1000 minutes
Links
- Introduction to optical microscopy . Photobleaching article (in English)
- Viegas MS, Martins TC, Seco F., Do Carmo A. Formalinfixed paraffin-embedded tissues (English) // Eur J Histochem: journal. - 2007. - Vol. 51 , no. 1 . - P. 59-66 . - PMID 17548270 .