To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.
Alena Rudkouskaya, Nattawut Sinsuebphon, Xavier Intes, Joseph E. Mazurkiewicz, and Margarida Barroso, "Fluorescence lifetime FRET imaging of receptor-ligand complexes in tumor cells in vitro and in vivo," Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 1006917 (Presented at SPIE BiOS: January 31, 2017; Published: 21 February 2017); https://doi.org/10.1117/12.2258231.
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