In recent years we have witnessed a rapid growth of the applications of fluorescence, and the development of novel measurement methods. One area of rapid growth has been in two-photon excitation, which is now practical due to the increasing availability of ps and fs lasers. In the present paper, we will show that using the fundamental output of a fs Titanium:Sapphire laser, it is possible and practical to observe three-photon excitation of DNA stains, Ca2+ probe Indo-1, or the intrinsic tryptophan fluorescence of proteins. Most studies of two-photon excitation use two photons of the same wavelength. We now showthat two-photon excitation can be obtained using two-photons at different wavelengths. The potential advantages of two-color two-photon excitation include localization of the excited volume at the region of beam overlap, and the possibility of increased selectivity by independent control of each laser beam. Another experimental opportunity is provided by the increasing availability of multiwavelength laser sources, which allow fluorescence experiments with multiple pulses. We used the phenomenon of stimulated emission to quench and modify the excited state populations. Light quenching allows selective removal of excited state fluorophores based on emission wavelength, decay time or orientation. In the case of evanescent waves due to total internal reflection (TIR) we show that light quenching can selectively remove fluorophores from the interface region, and provide spatially localized excitation 5000 Angstrom into the aqueous phase. And finally, we derive the development of metal-ligand complex probes which provide the opportunity to measure dynamics on the microsecond timescale. This versatile class of fluorophores allows a wide range of decay times and emission wavelengths based on the choice of ligand and metal. Importantly, transition metal-ligand complexes with non-identical dimine ligands display high fundamental anisotropies. Conjugatable MLCs have already been developed and used to measure correlation times as long as 5 microseconds.
