Quantitative spectral Linear Unmixing and Ratiometric FRET for live-cell imaging of protein interactions

We present a biophysical imaging strategy based on linear unmixing Förster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, we examined receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors. Ratiometric FRET measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, we explored physiological interactions between CD44 and 5-HT receptors and characterized the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis. Our findings demonstrate the utility of lux-FRET combined with quantitative molecular microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics.