RT Journal Article SR Electronic T1 Cytosolic Enhanced Dark Epac-Based FRET Sensors for Intracellular cAMP detection in live cells via FLIM JF bioRxiv FD Cold Spring Harbor Laboratory SP 2024.09.30.615785 DO 10.1101/2024.09.30.615785 A1 Zanetti, Giulia A1 Klarenbeek, Jeffrey B. A1 Jalink, Kees YR 2024 UL http://biorxiv.org/content/early/2024/10/01/2024.09.30.615785.abstract AB 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.Competing Interest StatementThe authors have declared no competing interest.