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

Summary

We present a protocol for quantitative analysis of molecules or ions in subcellular compartments of live cells using Förster resonance energy transfer (FRET)-based biosensors. It includes standardized imaging, binary mask processing, and ratiometric analysis with linear regression applied to phosphate in Arabidopsis thaliana (A. thaliana) but is broadly applicable to other biosensors and organisms.

Abstract

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.

Introduction

Monitoring metabolite and ion dynamics in live plant cells is essential for understanding how metabolic pathways and signaling networks are coordinated in space and time to support growth and development^1,2. Cellular metabolite levels fluctuate rapidly in response to environmental cues such as nutrient availability, light intensity, and stress³. Among these, the level of inorganic phosphate (Pi) is particularly important with key roles in energy transfer, signaling, and metabolic regulation, yet its subcellular dynamics remain difficult to measure⁴.

Protocol

NOTE: Arabidopsis transgenic lines expressing cpFLIPPi-5.3m and cpFLIPPi-null (cytosolic and plastid-targeted variants) were generated previously^17,18, and pht2;1-1 (CS19883) was obtained from the Arabidopsis Biological Resource Center, MS medium, cover glasses, perfluorodecalin, confocal microscope and a 40x oil-immersion objective (numerical aperture 1.3), excitation lasers (CFP/445 nm and cpVenus/515 nm or instrument-appropriate emission wavelengths), emission filters for CFP, cpVenus/FRET, and cpVenus acceptor, camera or detectors, image analysis workstation, FIJI²⁴ and Excel.

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Results

Confocal imaging

Using confocal microscopy and Metamorph software, separate channels were acquired to visualize the FRET donor and acceptor signals in A.thaliana leaf tissues of wildtype (WT) and the pht2;1 chloroplast Pi transporter mutant (Figure 2). Representative images illustrate clear subcellular localization of the Pi biosensor, with chloroplasts readily distinguishable not only within individual cells but also between dif.......

Discussion

This protocol provides a standardized approach for live-cell quantification of metabolites or ions using FRET-based biosensors. Key advantages include non-invasive ratiometric measurement and applicability to multiple subcellular compartments^17,20,21,22. The use of binary masks and linear regression improves precision and reproducibility. While demonstrated here for subcellular Pi in A. tha.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
40X oil-immersion objective	Olympus	https://www.edmundoptics.in/p/olympus-uplxapo-40x-oil-immersion-objective/55558/?srsltid=AfmBOoo0cqmgz7SoQ3ZCUvlJn_LeA37O42aX5BbFqF76k40CwQLNVn4P	Objective lens for imaging
Cover glasses / coverslips	VWR	48393-081	Cover slip for sample placement
FIJI (ImageJ)	Open source	2.9.0	Image analysis software
Inverted Olympus IX81 microscope	Olympus	http://www.olympusconfocal.com/brochures/pdfs/ix81.pdf	Confocal imaging system
iXon3 897 EMCCD camera	Andor Technology	https://andor.oxinst.com/products/ixon-emccd-camera-series/ixon-life-897	Part of confocal imaging system
MetaMorph software V7	Molecular Devices .LLC	Version 7	Image analysis workstation
Microsoft Excel	Microsoft Suite	v16.68	Data analysis software
Murashige & Skoog (MS) medium	Caisson	MSP11-10LT	Plant growth medium
Perfluorodecalin (Acros Organic)	Acros Organics	P9900-25G	Imaging medium
Phosphinothricin	Sigma	77182-82-2	Antibiotic
Plant growth chamber	Conviron	Custom	Growth chamber
Yokogawa CSU-X1 spinning disk confocal unit	Yokogawa	https://www.yokogawa.com/solutions/products-and-services/life-science/spinning-disk-confocal/csu-x1-confocal-scanner-unit/	Part of confocal imaging system