Quantitative Ratiometric Analysis of FRET-Based Biosensors in <em>Arabidopsis thaliana</em> Enables Live Measurement of Analytes in Subcellular Compartments

Fluorescent biosensors provide a non-invasive and versatile approach to monitor dynamic changes in metabolite or ion concentrations within live cells. Specifically, FRET-based biosensors enable ratiometric measurements that report subcellular analyte levels while being independent of biosensor expression levels. We describe a comprehensive and standardized protocol for the quantitative ratiometric analysis of FRET-based biosensors in plants. The protocol guides users through live-sample preparation, confocal image acquisition of donors, FRET, and acceptor channels, binary mask generation for subcellular regions of interest, followed by regression-based ratiometric data analysis. The primary output is regression-derived ratiometric readout that enables quantitative comparisons between genotypes, tissues, developmental stages, and treatment conditions. Using the cpFLIPPi-5.3m biosensor for inorganic phosphate as an example, we demonstrate measurement of inorganic phosphate levels in the chloroplast stroma of A. thaliana. This analytical framework is broadly applicable to other FRET-based biosensors and model systems, enabling precise spatiotemporal quantification of metabolites and ions in vivo. This strategy delivers measurable insights into the subcellular dynamics of metabolites and ions, supporting comparisons under varied experimental settings.