Lanthanides can be used because their small size (ionic radius) gives them the ability to replace metal ions inside protein complex such as calcium or nickel. The optical properties of lanthanide ions such as Ln(III) originate in the special features of their electronic [Xe]4f configurations. These configurations generate many electronic levels, the number of which is given by [14!/n!(14- n)!], translating into 3003 energy levels for Eu(III) and Tb(III).
The energies of these levels are well defined due to the shielding of the 4f orbitals by the filled 5s and 5p sub-shells, and are not very sensitive to the chemical environments in which the lanthanide ions are inserted. Inner-shell 4f-4f transitions span both the visible and near-infrared ranges. They are sharp and easily recognizable. Since these transitions are parity forbidden, the lifetimes of the excited states are long, which allows the use of time resolved spectroscopy, a definitive asset for bioassays and microscopy. The only drawback of f-f transitions are their faint oscillator strengths which may in fact be turned into an advantage.
The energy absorbed by the organic receptor (ligand) is transferred onto Ln(III) excited states, and sharp emission bands originating from the metal ion are detected after rapid internal conversion to the emitting level. The phenomenon is termed sensitization of the metal centered complex (also referred to as antenna effect) and is quite complex.
The energy migration path though goes through the long-lived triplet state of the ligand. Ln(III) ions are good quenchers of triplet states so that photobleaching is substantially reduced. The three types of transitions seen for lanthanide probes are: LMCT, 4f-5d, and intraconfigurational 4f-4f. The former two usually occur at energies too high to be relevant for bio-applications.