One vs Two-photon Excitation

In 1931, it was predicted theoretically by Maria Göppert-Mayer[1] that photons of lesser energy together can cause an excitation "normally" produced by the absorption of a single photon of higher energy in a process called two-photon excitation. If the excited molecule is fluorescent, it can emit a single photon of fluorescence as if it were excited by a single higher energy photon (see photos below). Since its first demostration by Webb et al in 1990, two-photon microscopy has been applied to a variety of imaging tasks and has now become the technique of choice for fluorescence microscopy in thick tissue and in live animals[2]. The Z-scan technique pioneered by Prof. Eric W. Van Stryland and Prof. David J. Hagan is the standard method to measure nonlinear absorption[3].

References:

[1] M. Göppert-Mayer, Ann. Phys., 1931, 9, 273-294.
[2] W. R. Zipfel, R. M. Williams, W. W. Webb, Nat. Biotechnol., 2003, 21, 1369-1377.
[3] M. Sheik-Bahea, A. A. Said, T. H. Wei, D. J. Hagan, E. W. Van Stryland, IEEE J. Quantum Electronics, 1990, 26, 760-769.

Below are demonstrations performed in our laboratory that show the high localization of two-photon excitation.

Experimental description:
The light from a tunable Coherent Mira 900 Ti:Sapphire laser (tuned to 760 nm, 200 fs pulse width, 76 MHz repetition rate) was equally split into two beams. A second-harmonic generator (APE, Germany) was used to double the frequency of one beam, which was used as the one-photon excitation source. The fundamental and second-harmonic beams were both expanded to be about 12 mm in diameter and then focused into a 1cm quartz cuvette by two 10x (UPLANFLN 10x/0.30, Olympus) objective lenses in opposite directions. A two-photon dye with a concentration of 5*10-5 M was pre-loaded inside the cuvette. [By Dr. Zhen-Li Huang, University of Central Florida]

Results: