Navigating FRET Imaging Techniques for Biosensor Applications

This tutorial paper is designed for biologists and bioscientists who want to integrate Förster Resonance Energy Transfer (FRET) in their research. It provides a bird’s eye view of the current FRET landscape, covering the factors that determine its occurrence and its effect on both the donor and acceptor fluorescence signal. This tutorial also provides practical guidance on the various methods used to measure the FRET phenomenon, including intensity-based FRET techniques, such as acceptor photobleaching and ratiometric FRET, as well as FLIM-based techniques, including fitting strategies and the phasor approach. Each technique is discussed in terms of its advantages and disadvantages, equipping researchers with the knowledge to select the most suitable approach for their objectives. Additionally, we present an overview for multiplexed FRET imaging and discuss its current potential and availability. The software packages available for analyzing FRET are also presented. This tutorial concludes with a discussion on the future of FRET experiments and the importance of improved hardware to support FRET biosensor readouts. Through this paper, biologists and bioscientists will gain a comprehensive understanding of FRET, its underlying principles, and the different techniques available for its quantification. Practical insights are shared to navigate the common obstacles and nuances of experimental design. This knowledge will facilitate the effective incorporation of FRET-based sensors into their research.