ABSTRACT

Maria Göppert-Mayer rst predicted the possibility of multiphoton excitation process in her doctoral dissertation presented in Göttingen.1 Experimental verication of multiphoton processes was not realized until 1963 when Kaiser and Garret rst observed two-photon excitation of CaF2:Eu2+ uorescence.2 Later, Kaiser and Garret further demonstrated two-photon excitation uorescence from organic molecules. ree-photon excitation was subsequently reported by Singh and Bradley.3 Multiphoton excitation of biochemical molecules provides complementary spectroscopic information to standard one-photon studies.4-7

e utilization of nonlinear optical processes to provide image contrast for microscopic studies was realized by pioneers such as Freund, Hellwarth, and Christensen.8 Nonlinear optical imaging has inherent 3-D resolution, which was rst suggested by the Oxford group including Sheppard et al.9,10 ey predicted that optical emissions that have quadratic or higher-order dependence on the excitation power will be conned to the focal plane of the microscope objective. e utilization of this principle for 3-D imaging was realized in the seminal work of Denk et al.11 Denk and coworkers further demonstrated the importance of this approach for limiting the photodamage and for extending the penetration depth in biological specimens. is work revolutionized the application of 3-D microscopy in biomedical imaging. e applications of other nonlinear optical mechanisms in biological imaging, such as second-and third-harmonic generation, sum frequencies generation, and coherent anti-Stokes Raman scattering, soon followed.12-14 is chapter will focus on multiphoton uorescence microscopy, the most widely used nonlinear microscopy approach, and its applications in biology and medicine.