Cytosolic‐enhanced dark Epac‐based <scp>FRET</scp> sensors allow for intracellular <scp>cAMP</scp> detection in live cells via <scp>FLIM</scp>

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 fluorophore's 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.