Chemical two-photon fluorescence.

ABSTRACT

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.