Cytosolic Enhanced Dark Epac-Based FRET Sensors for Intracellular cAMP detection in live cells via FLIM

Fluorescence Resonance Energy Transfer (FRET)-based biosensors are powerful tools for studying second messengers with high temporal and spatial resolution. FRET is commonly detected by ratio-imaging, but Fluorescence Lifetime Imaging Microscopy (FLIM), which measures the donor fluorophores lifetime, offers a robust and more quantitative alternative. We have introduced and optimized four generations of FRET sensors for cAMP, based on the effector molecule Epac1, including variants for either ratio-imaging or FLIM detection. Recently, Massengill and colleagues introduced additional mutations that improve cytosolic localization in these sensors, focusing on constructs optimized for ratio-imaging. Here we present and briefly characterize these mutations in our dedicated FLIM sensors, finding they enhance cytosolic localization while maintaining performance comparable to original constructs.