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optics Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultraviole ...
, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a
fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
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. 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 Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
, 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 Time-lapse microscopy is time-lapse photography applied to microscopy. Microscope image sequences are recorded and then viewed at a greater speed to give an accelerated view of the microscopic process. Before the introduction of the video tape ...
. However, photobleaching may also be used prior to applying the (primarily
antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
-linked) fluorescent molecules, in an attempt to quench
autofluorescence Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores) ...
. This can help improve the
signal-to-noise ratio Signal-to-noise ratio (SNR or S/N) is a measure used in science and engineering that compares the level of a desired signal to the level of background noise. SNR is defined as the ratio of signal power to the noise power, often expressed in deci ...
. 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 Photon energy is the energy carried by a single photon. The amount of energy is directly proportional to the photon's electromagnetic frequency and thus, equivalently, is inversely proportional to the wavelength. The higher the photon's frequency, ...
of the input light, or by employing more robust fluorophores that are less prone to bleaching (e.g. Cyanine Dyes,
Alexa Fluor The Alexa Fluor family of fluorescent dyes is a series of dyes invented by Molecular Probes, now a part of Thermo Fisher Scientific, and sold under the Invitrogen brand name. Alexa Fluor dyes are frequently used as cell and tissue labels in fluore ...
s or
DyLight Fluor The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescenc ...
s, 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 Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. ...
. 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 The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...
: 104–105 photons; 0.1–1.0 second lifetime. *Typical organic dye: 105–106 photons; 1–10 second lifetime. *CdSe/ZnS
quantum dot Quantum dots (QDs) are semiconductor particles a few nanometres in size, having light, optical and electronics, electronic properties that differ from those of larger particles as a result of quantum mechanics. They are a central topic in nanote ...
: 108 photons; > 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

*
Ozone depletion Ozone depletion consists of two related events observed since the late 1970s: a steady lowering of about four percent in the total amount of ozone in Earth's atmosphere, and a much larger springtime decrease in stratospheric ozone (the ozone l ...


References


External links


Introduction to Optical Microscopy
an article about photobleaching *{{cite journal , author=Viegas MS , author2=Martins TC , author3=Seco F , author4=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 , date=2007 , pmid=17548270 Microscopy Fluorescence Cell imaging Cell biology Articles containing video clips