Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells

We previously developed a technique known as homogeneous time-resolved fluorescence (HTRF) which has become a reference method in the design of bioassays. The technique makes use of the fluorescence resonance energy transfer (FRET) depending of the close proximity between europium cryptate, as a long-lived luminescent donor, and a matching fluorescent acceptor. The extension of this technique to in vivo cellular imaging is possible by the labeling of specific molecules within a living cell, each labeled either with the donor or acceptor and taking images of fluorescence resonance energy transfer (FRET). The time-resolved mode allows to discriminate the signal arising from molecules in close proximity from the background fluorescence of excess amounts of unbound fluorescent marker and from cell autofluorescence. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay upon direct excitation. The decay is also much slower than cell autofluorescence, and interference of cell autofluorescence is reduced under a time-resolved fluorescence microscope with a time-gated function equipped camera. The technique was evaluated both under a time-resolved fluorescence microscope and a microplate TRF reader.