The Tb(III) complex of the iron-transport protein transferrin (Tb_2-Tfr) exhibits strongly sensitised, sharp line luminescence from f-f states following multiphoton excitation via two tyrosinate residues directly co-ordinated to the lanthanide ion. Using an ultrafast Ti:sapphire laser system, a quadratic dependence of the Tb(III) luminescence intensity was observed on excitation with photons at 503 and 566 nm, and a cubic dependence with photons at 800 nm. The two-photon cross-sections at 503 and 566 nm are 7.4 * 10~(-50) and 0.37 * 10~(-50) cm~4 s photon~(-1) mol~(-1), respectively, which compare favourably with values reported for the green fluorescent protein. Three-photon excitation at 800 nm gives rise to a Tb(III) emission spectrum with excellent signal to noise ratios. These results lead to a proposal that if a Tb(III)-protein complex with similar luminescent properties could be formed in vivo, an intra-cellular imaging system that uses multiphoton-excited, long-lived lanthanide ion luminescence could be developed. This offers the prospect of multiphoton imaging in tight focal planes using sharp line emission with long lifetimes for wavelength and time discrimination against background fluorescence.
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