Using Multiphoton Microscopy for the Study of Embryogenesis

Abstract

Multiphoton fluorescence excitation imaging is an optical sectioning technique for fluorescence microscopy. At very high photon densities, two or more photons may coordinately excite an energy transition in a fluorophore that corresponds to the sum of the energies of the individual photons. by this means, a fluorophore may be excited by a wavelength that is considerably longer than its single photon excitation wavelength. Ultra-fast pulsed (femtosecond) lasers can produce the peak power densities in the focal volume of an objective lens needed to provide sufficient 2- or 3- photon excitation events for imaging. The use of short-pulse lasers provides the high peak powers necessary for imaging yet with modest mean power levels that do not thermally damage biological specimens. Production of multiphoton events depends on the square of photon density for 2-photon excitation and the cube of photon density for 3-photon excitation. The power density therefore rapidly falls off away from the focal volume of an objective lens, thereby confining fluorescence excitation to the focal volume.