Two-photon excitation microscopy

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 can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection and reduced phototoxicity.[1]

Two-photon excitation employs a concept first described by Maria Goeppert-Mayer (1906-1972) in her 1931 doctoral dissertation.[2], and first observed in 1962 in cesium vapor using laser excitation by Isaac Abella [3]

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.[4] 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 patented by Winfried Denk, James Strickler and Watt Webb at Cornell University.[5] Carl Zeiss currently holds this patent; Olympus Inc. has licensed it to sell 2-photon microscopes.

A diagram of a two-photon microscope

The most commonly used fluorophores have excitation spectra in the 400–500 nm range, whereas the laser used to excite the fluorophores 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 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, 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 use of infrared light to excite fluorophores in light-scattering tissue has added benefits.[6] 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 the focal volume. Compared to a confocal microscope, photon detection is much more effective since even scattered photons contribute to the usable signal. There are several caveats to using two-photon microscopy: The pulsed lasers needed for two-photon excitation are much more expensive then the constant wave (CW) lasers used in confocal microscopy. The two-photon absorption spectrum of a molecule may vary significantly from its one-photon counterpart. For very thin objects such as isolated cells, single-photon (confocal) microscopes can produce images with higher optical resolution due to their shorter excitation wavelengths. In scattering tissue, on the other hand, the superior optical sectioning and light detection capabilities of the two-photon microscope result in better performance.

Contents

Higher-order excitation

Simultaneous absorption of three or more photons is also possible, allowing for three-photon or multiphoton excitation microscopy.

See also

References

  1. ^ Denk W, Strickler J, Webb W (1990). "Two-photon laser scanning fluorescence microscopy". Science 248 (4951): 73–6. doi:10.1126/science.2321027. PMID 2321027. 
  2. ^ Goeppert-Mayer M (1931). "Über Elementarakte mit zwei Quantensprüngen". Ann Phys 9: 273–95. doi:10.1002/andp.19314010303. http://adsabs.harvard.edu/abs/1931AnP...401..273G. 
  3. ^ I. D. Abella (1962) "Optical Double-Photon Absorption in Cesium Vapor," Phys. Rev. Letters. 9, 453.
  4. ^ Denk W, Svoboda K (1997). "Photon upmanship: why multiphoton imaging is more than a gimmick". Neuron 18 (3): 351–7. doi:10.1016/S0896-6273(00)81237-4. PMID 9115730. 
  5. ^ US patent 5034613 "Two-photon laser microscopy."
  6. ^ Helmchen F, Denk W (2005). "Deep tissue two-photon microscopy". Nat Methods 2 (12): 932–40. doi:10.1038/nmeth818. PMID 16299478. 

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