Two-photon excitation microscopy

From ArticleWorld

Two-photon excitation microscopy is a method that allows the imaging of living tissue. This method is able to capture images up to one millimeter in depth and has several advantages over confocal microscopy. The two major advantages are the ability to get images through deeper tissue penetration that may not be captured by confocal microscopy techniques as well as the reduced phototoxicity of the method.

History

This concept was first introduced by Maria Göppert-Mayer in her doctoral dissertation in 1931. The concept was put fought based on the idea that two photons of low energy can cause excitation of a fluorophore and result in the emission of fluorescene. In order to initiate simultaneous absorption of the photons however a high flux of excitation photons would be required as the probability of simultaneous absorption is low otherwise.

This original concept was pioneered by Winfried Denk of Cornell University. He combined this concept with the use of a laser scanner. Today this method uses an infrared laser beam that is focused through an objective lens. The beam used laser used can normally allow high proton density and flux to enable simultaneous photon absorption over a wide range of wavelengths.

Lasers

The most common fluorophores can be stimulated to absorb two photons simultaneouesly by the use of the proper laser. If this occurs then the fluorospore will be elevated to the next excitation level and will emit a single photon. The use of infrared lasers to excite fluoropores in light-scattering tissues has many benefits. These are that long wavelengths are scatterred less and this leads to better resolution also lower-energy photons are less likely to cause damage outside of the focal volume.

The disavantages of this type of microscopy are that the lasers are ususally more expensive , the microscope requires special modifications to avoid damage due to the intense pulses and that wavelengths may be absorbed by the water in the living tissues.