In our research, we use macroscopic fluorescence lifetime imaging (MFLI) to measure Fӧrster resonance energy transfer (FRET) between receptor-antibody complexes and evaluate the binding of therapeutic antibodies to tumor xenografts. In the cover image of the June 14 issue of Biophysical Reports, we have compared MFLI-based FRET with intensity-based FRET to measure FRET between intravenously injected transferrin labeled with two near-infrared dyes upon binding of transferrin to its dimeric receptor in the liver and the bladder.

The image shows three mice observed in three spectral channels (one channel per row), with two of the mice being used only as references to extract the relevant information from the mouse of interest (the rightmost mouse). A (deleted) combination of these nine pieces of data yields the sought-for information, namely the degree of ligand-to-target interaction in the two organs of interest: liver (bright center area in the rightmost mouse) and bladder (bright region at the bottom of the same mouse).

The raw images shown here were collected by using an in vivo imaging system optical imager and are further analyzed to extract mean intensity-based FRET, as described in the paper. The observation is that FRET is observed between transferrin/transferrin receptor complexes in the liver but not in the bladder, consistent with transferrin receptors being highly expressed in the liver.

The research shown in this article indicates that FRET can be measured by using intensity-based imaging of intact, live mice, although with a fairly large uncertainty, while MFLI-FRET measurements are much simpler and more reliable, allowing researchers to measure the binding of near-infrared-labeled ligands injected into the bloodstream of mice to their respective receptors. You can find more information on our work at https://sites.google.com/view/barrosolab/home and https://intes-lab.bme.rpi.edu/.

— Jason T. Smith, Nattawut Sinsuebphon, Alena Rudkouskaya, Xavier Michalet, Xavier Intes, and Margarida Barroso