Two-photon fluorescent labels with enhanced sensitivity for biological imaging

The development of two-photon laser scanning fluorescence microscopy (TPLSM) has provided a new capability for 3D imaging in whole and living tissues, reduced photobleaching effects, and greater depth penetration. Since the rate of two-photon excitation is proportional to the square of the light intensity, degree of excitation falls off roughly as the fourth power of distance from the focal plane. The full potential of two-photon fluorescence microscopy has not been realized, in part, because fluorophores under use have been developed for single photon excitation and have not been optimized for two-photon absorption. Highly efficient two-photon fluorophores are needed to allow sensitive detection of small numbers of labeled sites and to reduce the optical power of the exciting laser beam that is needed to produce adequate signal levels. The sensitivity of a two-photon fluorescence label is determined by the product of the two-photon absorption cross section and the fluorescence quantum efficiency.