From principles to practice: a comprehensive guide to FRET-FLIM in plants

Abstract Förster Resonance Energy Transfer combined with Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) is revolutionizing plant biology, by enabling the study of protein–protein interactions (PPIs) within live cells. This manuscript describes the principles of FRET and the practical application of FRET acceptor photobleaching (FRET-APB) in exploring PPIs in vivo . It mainly focuses on the superior characteristics of FRET-FLIM and details the materials and methods for implementing this technique in plants. It provides a profound overview about the required instruments, protocols for sample preparation, methods for calibration and acquisition, and pipelines for data analyses including novel analyses for binding and FRET efficiencies. Furthermore, it discusses the potential pitfalls and challenges related to the sample autofluorescence, protein expression heterogeneity, and acquisition photodamage or bleaching. This works aims to highlight the great prospects of FRET-FLIM in advancing our understanding of PPIs in living plant cells.