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. LAY DESCRIPTION: All biological systems are made up, in essence, of molecules interacting with another. These can be proteins interacting with proteins, DNA or RNA interacting with each other or with proteins, lipids, sugars or other small molecules. While there are many well described methods for studying intermolecular interactions in test tubes and in cells, it is more challenging to apply these to model organisms such as zebrafish, Xenopus and mice. This is because the samples are thicker, requiring optics with a long depth of field and sensitive detectors since signals from fluorescent proteins can be scattered inside the tissues of these systems. FRET is the non‐radiative energy transfer from an excited donor fluorophore to an acceptor when donor and acceptor are within 1–10 nm of one another – the distance expected between interacting proteins. FRET results in decreased donor fluorescence intensity and lifetime (time spent in exited state). Potential interaction partners are therefore tagged with donor and acceptor fluorophores to measure this interaction by FRET. The extent of interaction (how many donor molecules interact, how strongly) and the distance between interaction partners affect the efficiency of FRET (E = 1 – IDA/ID). We chose FRET as a useful fluorescence microscopy method to study interactions in early zebrafish embryos. This is because it can be assessed using different techniques and does not necessarily require complex mathematical analysis, such as regression and curve fitting. Measuring FRET by Fluorescence Lifetime Imaging Microscopy (FRET‐FLIM) is more robust to photobleaching and changes in relative concentrations than intensity‐based measurements. FLIM‐FRET is a type of FRET where the donor lifetime is measured in presence and absence of the acceptor. Traditional methods of assessing FLIM rely on accurate measurements with a high signal‐to‐noise ratio (SNR), and intensive analysis. Experiments in live 3D samples have been, up until now, difficult as signal is lower and acquisition times are limited because some biological processes happen more quickly than the time needed to obtain FLIM data. Here we applied a newer method of FLIM‐FRET termed Average Arrival Time FRET. It does not require fitting, is comparatively fast, gentle and sensitive so providing accessible, quantitative lifetime‐based information for those interested in using FRET methods to study molecular interactions in real time and 3D in model organisms such as zebrafish. [ABSTRACT FROM AUTHOR]