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Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one millimetre in depth. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced phototoxicity. ==Concept== Two-photon excitation employs two-photon absorption, a concept first described by Maria Goeppert-Mayer (1906–1972) in her doctoral dissertation in 1931,〔 〕 and first observed in 1961 in a CaF2:Eu2+ crystal using laser excitation by Wolfgang Kaiser.〔 〕 Isaac Abella showed in 1962 in cesium vapor that two-photon excitation of single atoms is possible.〔 〕 The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event. Each photon carries approximately half the energy necessary to excite the molecule. An excitation results in the subsequent 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 from a femtosecond laser. The purpose of employing the two-photon effect is that the axial spread of the point spread function is substantially lower than for single-photon excitation. As a result, the resolution along the z dimension is improved, allowing for thin optical sections to be cut. In addition, in many interesting cases the shape of the spot and its size can be designed to realize specific desired goals. Two-photon microscopes are less damaging to the sample than a single-photon confocal microscope. The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the two-photon fluorescence lies in the ~700–1000 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 are absorbed during the excitation of the 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, excitation is restricted to the tiny focal volume (~1 femtoliter), resulting in a high degree of rejection of out-of-focus objects. This ''localization of excitation'' is the key advantage compared to single-photon excitation microscopes, which need to employ additional elements such as pinholes to reject out-of-focus fluorescence. 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 scan head is typically composed of two mirrors, the angles of which can be rapidly altered with a galvanometer. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Two-photon excitation microscopy」の詳細全文を読む スポンサード リンク
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