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-selective 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, also called pyranine. This approach addresses the limitations of genetic sensors and traditional pH measurement techniques by offering a nontoxic, cost-effective and precise method for pH assessment at cellular resolution via ratiometric confocal imaging. In addition, we introduce an updated Fiji plugin for ratiometric image conversion. The new plugin enhances workflow efficiency by automating image processing while offering several options for customization, thereby ensuring reliable and reproducible results. The full procedure, from staining to image analysis, can be completed within ~2-4 h, depending on the number of samples and imaging depth. This protocol provides a robust tool for plant physiologists to investigate apoplastic pH dynamics with high spatial resolution and accuracy in plant tissues.