Multiphoton Excitation of Biochemical Fluorophores

Abstract

Pulsed lasers are used in many laboratories as an excitation source for timeresolved fluorescence spectroscopy. Although these laser sources are most often used for one-photon excitation (1PE), the high peak power of picosecond and femtosecond laser pulses can result in two-photon excitation (2PE), wherein the fluorophore simultaneously absorbs two long-wavelength photons to yield the first excited singlet state. Two-photon absorbance or excitation requires high peak powers to increase the probability that two photons are simultaneously available for absorption. Because of the interaction of two photons with the fluorophore, the selection rules for light absorption are, in principle, different from those for one-photon spectroscopy. Hence, two-photon spectroscopy was initially used as a tool to study the excited-state symmetry of organic chromophores or to identify additional energy levels. Two-photon spectroscopy has been applied to a wide range of chromophores, including the study of excited states of aromatic hydrocarbons, porphyrins, and polyenes. Two-photon excitation has also become a tool of the biophysical scientists, as evidenced by two-photon studies of protein-bound chromophores and indole derivatives. While polarized excitation is often used in two-photon spectroscopy, the experimental conditions and interpretation of the data are distinct from its use in time-resolved fluorescence. In classical two-photon spectroscopy one obtains information about the symmetry of the excited state from the differential absorption of linearly and circularly polarized light of molecules in fluid solution.