Chemical Two-Photon Fluorescence

We describe a method based on a caged fluorescent molecule that can act as a chemical two-photon probe. It is composed of an organic fluorophore and a ruthenium bipyridine complex that acts as a photoremovable quencher. For the fluorophore to be emissive, two independent photons must act on the molecule: the first photon frees the fluorescent ligand from the Ru complex and the second photon excites the fluorescence. In this two-photon regime, the emission is not proportional to the excitation intensity but rather to its second power, as in traditional two-photon systems based on ultrashort pulsed high-power lasers. This quadratic relationship implies a much higher spatial precision on the z-axis when the probe is used in a microscopy technique. The chemical nature of the two-photon excitation mechanism allows the use of inexpensive low-power lasers.