Two-photon excitation microscopy 2105059 221207458 2008-06-23T14:45:08Z DOI bot 6652755 Citation maintenance. You can [[WP:DOI|use this bot]] yourself! Please [[User:DOI_bot/bugs|report any bugs]]. '''Two-photon excitation microscopy''' is a [[fluorescence]] imaging technique that allows imaging living tissue up to a depth of one millimeter. The two-photon excitation [[microscope]] is a special variant of the [[multiphoton fluorescence microscope]]. Two-photon excitation may in some cases be a viable alternative to [[confocal microscopy]] due to its deeper tissue penetration and reduced [[phototoxicity]].<ref name=Denk_1990>{{cite journal |author=Denk W, Strickler J, Webb W |title=Two-photon laser scanning fluorescence microscopy |journal=Science |volume=248 |issue=4951 |pages=73–6 |year=1990 |pmid=2321027 |doi=10.1126/science.2321027}}</ref> Two-photon excitation employs a concept first described by [[Maria Göppert-Mayer]] ([[1906]]-[[1972]]) in her 1931 doctoral dissertation.<ref name=Göppert-Mayer_1931>{{cite journal | author =Göppert-Mayer M | title = Über Elementarakte mit zwei Quantensprüngen | journal = Ann Phys | year = 1931 | volume = 9| pages = 273&ndash;95 | url=http://adsabs.harvard.edu/abs/1931AnP...401..273G | doi = 10.1002/andp.19314010303 }}</ref>, and first observed in 1962 in cesium vapor using laser excitation by [[Isaac Abella]] <ref> I. D. Abella (1962) "Optical Double-Photon Absorption in Cesium Vapor," Phys. Rev. Letters. '''9''', 453. </ref> The concept of two-photon excitation is based on the idea that two photons of low energy can excite a [[fluorophore]] in a quantum event, resulting in the emission of a fluorescence photon, typically at a higher energy than either of the two excitatory photons. The probability of the near-simultaneous absorption of two photons is extremely low. Therefore a high [[flux]] of excitation photons is typically required, usually a femtosecond [[laser]]. Two-photon [[microscopy]] was pioneered by [[Winfried Denk]] in the lab of [[Watt W. Webb]] at [[Cornell University]]. He combined the idea of two-photon absorption with the use of a laser scanner.<ref name=Denk_1997>{{cite journal |author=Denk W, Svoboda K |title=Photon upmanship: why multiphoton imaging is more than a gimmick |journal=Neuron |volume=18 |issue=3 |pages=351–7 |year=1997 |pmid=9115730 |doi=10.1016/S0896-6273(00)81237-4}}</ref> In two-photon excitation microscopy an infrared laser beam is focused through an objective lens. The [[Ti-sapphire laser]] normally used has a pulse width of approximately 100 femtoseconds and a repetition rate of about 80 MHz, allowing the high photon density and flux required for two photons absorption and is tunable across a wide range of wavelengths. Two-photon technology has been [[patent]]ed by Winfried Denk, James Strickler and Watt Webb at Cornell University.<ref>{{patent|US| 5034613|"Two-photon laser microscopy."}}</ref> Carl Zeiss currently holds this patent; Olympus Inc. has licensed it to sell 2-photon microscopes. [[Image:Diagram of a two-photon excitation microscope en.svg|thumb|300px|A diagram of a two-photon microscope]] The most commonly used fluorophores have excitation spectra in the 400&ndash;500 nm range, whereas the laser used to excite the fluorophores lies in the ~700&ndash;1000 nm (infrared) range. If the fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to be raised into the excited state. The fluorophore will then emit a single photon with a wavelength that depends on the type of fluorophore used (typically in the visible spectrum). Because two photons need to be absorbed to excite a fluorophore, the probability for fluorescent emission from the fluorophores increases quadratically with the excitation intensity. Therefore, much more two-photon fluorescence is generated where the laser beam is tightly focused than where it is more diffuse. Effectively, fluorescence is observed in any appreciable amount in the focal volume, resulting in a high degree of rejection of out-of-focus objects. The fluorescence from the sample is then collected by a high-sensitivity detector, such as a [[photomultiplier]] tube. This observed light intensity becomes one [[pixel]] in the eventual image; the focal point is scanned throughout a desired region of the sample to form all the pixels of the image. The use of infrared light to excite fluorophores in light-scattering tissue has added benefits.<ref name=Helmchen_2005>{{cite journal |author=Helmchen F, Denk W |title=Deep tissue two-photon microscopy |journal=Nat Methods |volume=2 |issue=12 |pages=932–40 |year=2005 |pmid=16299478 |doi=10.1038/nmeth818}}</ref> Longer wavelengths are scattered to a lesser degree than shorter ones, which is a benefit to high-resolution imaging. In addition, these lower-energy photons are less likely to cause damage outside of the focal volume. There are several caveats to using two-photon microscopy: Pulsed lasers are generally much more expensive, the microscope requires special optics to withstand the intense pulses, the two-photon absorption spectrum of a molecule may vary significantly from its one-photon counterpart, and wavelengths greater than 1400 nm may be significantly absorbed by the water in living tissue. == See also == * [[3D optical data storage]] ==References== <!-- ---------------------------------------------------------- See http://en.wikipedia.org/wiki/Wikipedia:Footnotes for a discussion of different citation methods and how to generate footnotes using the <ref>, </ref> and <reference /> tags ----------------------------------------------------------- --> {{reflist}} == External links == * [http://parkerlab.bio.uci.edu/attachments/build2photon/Sanderson_microscopy_article%5B1%5D.pdf Acquisition of Multiple Real-Time Images for Laser Scanning Microscopy] (Sanderson microscopy article) * [http://parkerlab.bio.uci.edu/microscopy_construction/build_your_own_twophoton_microscope.htm Build Your Own Video-Rate 2-photon Microscope] [[Category:Microscopes]] [[Category:Cell imaging]] [[de:Multiphotonenmikroskop]] [[es:Microscopía de excitación de dos fotones]] [[ja:2光子励起顕微鏡]] [[pl:Mikroskop dwufotonowy]] [[ru:Двухфотонный лазерный микроскоп]]