| Revision as of 13:34, 5 April 2011 editUnaUnsoS (talk | contribs)14 edits Included information about chemical strategies to retard photobleaching, with a reference.← Previous edit | Revision as of 17:28, 5 April 2011 edit undoSmokefoot (talk | contribs)Autopatrolled, Extended confirmed users, Pending changes reviewers, Rollbackers74,236 edits rv specialized refNext edit → | ||
| Line 9: | Line 9: | ||
| 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. ]s or ]s). 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. | 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. ]s or ]s). 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. | ||
| Chemically, different strategies have been proposed for how to improve the fluorescence rate and decrease photobleaching including deoxygenation and addition of ], ], ] ], or ] quenchers.<ref>{{cite journal |author=Jerker Widengren, Andriy Chmyrov, Christian Eggeling, Per-Åke Löfdahl, and Claus A. M. Seidel |year=2007 |title=Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy |journal=The Journal of Physical Chemistry A |volume=111 |issue=3 |pages=429–440 | doi=10.1021/jp0646325 | pmid=17228891 }}</ref> | |||
| ==Lifetime== | ==Lifetime== | ||
| Line 22: | Line 21: | ||
| * an article about photobleaching | * an article about photobleaching | ||
| *{{cite journal |author=Viegas MS, Martins TC, Seco F, do Carmo A |title=An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues |journal=Eur J Histochem |volume=51 |issue=1 |pages=59–66 |year=2007 |pmid=17548270 }} | *{{cite journal |author=Viegas MS, Martins TC, Seco F, do Carmo A |title=An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues |journal=Eur J Histochem |volume=51 |issue=1 |pages=59–66 |year=2007 |pmid=17548270 }} | ||
| ==References== | |||
| <references/> | |||
| ] | ] | ||
Revision as of 17:28, 5 April 2011
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, 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.
Lifetime
Depending on the material, dyes can produce different photon numbers and therefore have different lifetimes (at e.g. 10 photons/s):
- Green fluorescent protein: 10-10; 0.1-1 s
- Typical organic dye: 10-10; 1-10 s
- CdSe/ZnS Quantum dot: 10; > 1000 s
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.
{{cite journal}}: CS1 maint: multiple names: authors list (link)
 | This cell biology article is a stub. You can help Misplaced Pages by expanding it. |
 | This optics-related article is a stub. You can help Misplaced Pages by expanding it. |