In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules.[1][2] Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies. 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 improve the signal-to-noise ratio.
Photobleaching may also be exploited to study the motion and/or diffusion of molecules, for example via the FRAP, in which movement of cellular components can be confirmed by observing a recovery of fluorescence at the site of photobleaching, or FLIP techniques, in which multiple rounds of photobleaching is done so that the spread of fluorescence loss can be observed in cell.
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. Cyanine Dyes, Alexa Fluors or DyLight Fluors, AttoDyes, Janelia Dyes and others). 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.
Photobleaching is an important parameter to account for in real-time single-molecule fluorescence imaging in biophysics. At light intensities used in single-molecule fluorescence imaging (0.1-1 kW/cm2 in typical experimental setups), even most robust fluorophores continue to emit for up to 10 seconds before photobleaching in a single step. For some dyes, lifetimes can be prolonged 10-100 fold using oxygen scavenging systems (up to 1000 seconds with optimisation of imaging parameters and signal-to-noise). For example, a combination of Protocatechuic acid (PCA) and protocatechuate 3,4-dioxygenase (PCD) is often used as oxygen scavenging system, and that increases fluorescence lifetime by more than a minute.
Depending on their specific chemistry, molecules can photobleach after absorbing just a few photons, while more robust molecules can undergo many absorption/emission cycles before destruction:
^Demchenko, Alexander P (2020-02-20). "Photobleaching of organic fluorophores: quantitative characterization, mechanisms, protection". Methods and Applications in Fluorescence. 8 (2): 022001. doi:10.1088/2050-6120/ab7365. ISSN 2050-6120. PMID 32028269. S2CID 211048171.
^"Fluorophore Photobleaching Literature References".
External linksEdit
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.
October 16, 2023
photobleaching, optics, photobleaching, sometimes, termed, fading, photochemical, alteration, fluorophore, molecule, such, that, permanently, unable, fluoresce, this, caused, cleaving, covalent, bonds, specific, reactions, between, fluorophore, surrounding, mo. In optics photobleaching sometimes termed fading is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce This is caused by cleaving of covalent bonds or non specific reactions between the fluorophore and surrounding molecules 1 2 Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores The number of excitation cycles to achieve full bleaching varies 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 source 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 improve the signal to noise ratio Photobleaching may also be exploited to study the motion and or diffusion of molecules for example via the FRAP in which movement of cellular components can be confirmed by observing a recovery of fluorescence at the site of photobleaching or FLIP techniques in which multiple rounds of photobleaching is done so that the spread of fluorescence loss can be observed in cell 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 Cyanine Dyes Alexa Fluors or DyLight Fluors AttoDyes Janelia Dyes and others 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 Photobleaching is an important parameter to account for in real time single molecule fluorescence imaging in biophysics At light intensities used in single molecule fluorescence imaging 0 1 1 kW cm2 in typical experimental setups even most robust fluorophores continue to emit for up to 10 seconds before photobleaching in a single step For some dyes lifetimes can be prolonged 10 100 fold using oxygen scavenging systems up to 1000 seconds with optimisation of imaging parameters and signal to noise For example a combination of Protocatechuic acid PCA and protocatechuate 3 4 dioxygenase PCD is often used as oxygen scavenging system and that increases fluorescence lifetime by more than a minute Depending on their specific chemistry molecules can photobleach after absorbing just a few photons while more robust molecules can undergo many absorption emission cycles before destruction Green fluorescent protein 104 105 photons 0 1 1 0 second lifetime Typical organic dye 105 106 photons 1 10 second lifetime CdSe ZnS quantum dot 108 photons gt 1 000 seconds lifetime This use of the term lifetime is not to be confused with the lifetime measured by fluorescence lifetime imaging See also EditOzone depletionReferences Edit Demchenko Alexander P 2020 02 20 Photobleaching of organic fluorophores quantitative characterization mechanisms protection Methods and Applications in Fluorescence 8 2 022001 doi 10 1088 2050 6120 ab7365 ISSN 2050 6120 PMID 32028269 S2CID 211048171 Fluorophore Photobleaching Literature References External links EditIntroduction 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 Retrieved from https en wikipedia org w index php title Photobleaching amp oldid 1157440238, wikipedia, wiki, book, books, library,
