Quantitative imaging approaches for small-molecule measurements using FRET sensors in plants

Cellular metabolites and ions can exhibit very specific spatiotemporal dynamics that are very challenging to monitor using extraction-based methods. Genetically encoded Föster resonance energy transfer sensors afford a powerful method of measuring these dynamics in situ and hence are now widely used in order to decode information communicated through the dynamics of cellular metabolites and ions. This methodology involves (1) the development of a suitable sensor, (2) genetic engineering of the sensor for its expression in the tissue of interest, and (3) measurement and characterization of the cellular metabolites and ions using optical imaging. This chapter describes the measurement aspects. We describe the imaging setup, sample preparation from leaf discs and root cells, performance of a perfusion experiment, and quantification of metabolite and ion concentrations from the imaging data. We also describe post-experiment analysis including estimation of sensor efficiency and spectral bleedthrough.