Second Harmonic Generation Microscopy Versus Third Harmonic Generation Microscopy in Biological Tissues

Abstract

Second-harmonic generation (SHG) and third-harmonic generation (THG) processes are both nonlinear processes, related to the interaction of intense light with matters. SHG process describes the generation of light wave that is twice the frequency (with half of the original wavelength) of the original one while THG process describes the generation of light wave that triples the frequency (with one third of the original wavelength) of the original one. The harmonic light wave generation is coupled from the excited nonlinear polarization P ^NL under intense laser excitation. The interaction of nonlinear polarization P ^NL and the excitation light is usually related through a nonlinear susceptibility χ, as previously described in Chaps. 7 and 8. SHG and THG can be visualized by considering the interaction in terms of the exchange of photons between various frequencies of the fields. According to this picture, which is previously illustrated in Figs. 8.1 (a), (b), two or three photons of angular frequency ω are destroyed and a photon of angular frequency 2ω (for SHG) or 3ω (for THG) is created in a single quantum-mechanical process. The solid lines in the figure represent the atomic ground states, and the dashed lines represent what are known as virtual levels. These virtual levels are not energy eigenlevels of the atoms, but rather represent the combined energy of one of the energy eigenstates of the atom and one or more photons of the radiation field. Due to its virtual level transition characteristics, harmonic generations are known to leave no energy deposition to the interacted matters, since no real transition involved and the emitted photon energy will be exactly the same as the total absorbed photon energy. This virtual transition characteristic provides the optical “noninvasive” nature desirable for microscopy applications, especially for live biological imaging.