In Vivo Quantification of Intramolecular FRET Using RacFRET Biosensors

Genetically encoded FRET biosensors are powerful tools to visualize protein activity and signaling events in vivo. Compared with a biochemical approach, FRET biosensors allow a noninvasive spatial-temporal detection of signaling processes in live cells and animal tissues. While the concept of this technique is relatively simple, the experimental procedure is complicated and consists of several steps: (1) biosensor optimization; (2) data acquisition; and (3) image processing with each step posing its own challenge. In this chapter, we discuss steps (2) and (3) with the emphasis on the intramolecular RacFRET biosensor. We describe the design principle of the biosensor, the experimental imaging setup for acquiring FRET data in zebrafish embryos expressing the RacFRET biosensor, and the step-by-step ratio image generation protocol using Fiji software. We discuss important considerations during FRET data acquisition and analysis. Finally, we provide a macro code for the automated ratio image generation.