Multiphoton Microscopy

Authored by: Kert Edward

Handbook of Optical Sensors

Print publication date:  October  2014
Online publication date:  October  2014

Print ISBN: 9781439866856
eBook ISBN: 9781439866863
Adobe ISBN:


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Multiphoton microscopy (MPM) refers to a relatively new imaging technology whereby multiple photons are nearly simultaneously absorbed by a sample in the focal plane, resulting in the emission of a fluorescence signal. Since the probability of photon absorption is greatest in the excitation focal volume and negligibly small elsewhere, the procedure allows for inherent optical sectioning. This phenomenon was first predicted by Maria Göppert-Mayer in 1931 in her doctoral thesis (Göppert-Mayer 1931) but not verified until the 1960s by Franken et al. (1961) after the invention of the laser. This is due in par+t to the requirement of a very intense, temporally, and spatially confined coherent light source to maximize the likelihood of multiple photon absorption by a fluorophore (a molecule which fluoresces upon excitation). The most common example of a multiphoton system is the two-photon laser scanning fluorescence microscope that was first realized by Denk et al. (1990). Today, several commercial systems are available based on this initial prototype. In Figure 9.1, the LSM 7MP system from Zeiss is shown. In a typical system, a high power, high repetition rate (80–100 MHz) femtosecond laser is utilized in conjunction with a high numerical aperture (NA) objective, to achieve the required spatial and temporal photon confinement. This results in the near simultaneous absorption of two-photons in a subfemtoliter volume. Three-photon absorption is also possible; hence, the descriptive terminology of MPM is sometime used.

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