Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.