Quantitative imaging of apoplastic pH in plant roots via confocal microscopy.

The regulation of apoplastic pH is critical for plant growth and development, affecting processes such as nutrient uptake, cell wall expansion, and intercellular signaling. Conventional methods for measuring apoplastic pH, including pH indicators in growth media and ion-specific electrodes, often fall short of providing the spatial resolution and accuracy needed for detailed studies. Here, we present a protocol for the quantitative imaging of apoplastic pH in Arabidopsis thaliana roots using confocal microscopy combined with the fluorescent pH probe 8-hydroxy-pyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), also called pyranine. This approach addresses the limitations of genetic sensors and traditional pH measurement techniques by offering a non-toxic, cost-effective, and precise method for pH assessment at cellular resolution via ratiometric confocal imaging. Additionally, we introduce an updated Fiji plugin for ratiometric image conversion, which enhances workflow efficiency by automating image processing while offering several options for customization, thereby ensuring reliable and reproducible results. This protocol provides a robust tool for plant physiologists to investigate apoplastic pH dynamics with high spatial resolution and accuracy in plant tissues.