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Quantitative imaging of apoplastic pH in plant roots via confocal microscopy

Nature Protocols (2026)Cite this article

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Abstract

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.

Key points

  • This protocol enables 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.

  • 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 a cellular resolution via ratiometric confocal imaging.

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Fig. 1: HPTS.
Fig. 2: Workflow for HPTS staining of Arabidopsis roots.
Fig. 3: The ratiometric image conversion process.
Fig. 4: RALF1 treatment increases apoplastic pH.

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Data availability

All data supporting the findings of this study are available via Freidata at https://doi.org/10.60493/tjf4b-4jx39 (ref. 41). Source data are provided with this paper.

Code availability

The Fiji plugins developed for HPTS analysis and the Python script for generating user-specific calibration curves can be found via Zenodo: ratiometric image conversion plugin at https://doi.org/10.5281/zenodo.15658668 (ref. 36), Ratio2pH plugin at https://doi.org/10.5281/zenodo.15599805 (ref. 37) and Python script for 4PL calibration fitting at https://doi.org/10.5281/zenodo.17303477 (ref. 35). The workflow builds on earlier ratiometric imaging approaches (ref. 6 and https://doi.org/10.5281/zenodo.8387238)42.

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Acknowledgements

We thank the LIC Imaging Center Freiburg for expertise and support. This project is funded by the Deutsche Forschungsgemeinschaft (German Research Foundation): CIBSS—EXC2189 project ID 390939984 (to E.B.) and confocal microscopy project IDs 414136422 and 499026372.

Author information

Authors and Affiliations

  1. Institute of Molecular Plant Physiology, Faculty of Biology, University of Freiburg, Freiburg, Germany

    Ann-Kathrin Rößling, Niklas Mayle & Elke Barbez

  2. Center for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Ann-Kathrin Rößling & Elke Barbez

  3. Biozentrum, University of Basel, Basel, Switzerland

    Laurent Guerard

Authors
  1. Ann-Kathrin Rößling
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  2. Niklas Mayle
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Contributions

E.B. and A.-K.R. conceived the project and designed the experiments, A.-K.R. performed the experiments, N.M. and L.G. wrote and optimized the image conversion script, and E.B. and A.-K.R. wrote the paper with input from all authors.

Corresponding author

Correspondence to Elke Barbez.

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Nature Protocols thanks Quang Tran and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Barbez, E. et al. Proc. Natl Acad. Sci. USA 114, E4884–E4893 (2017): https://doi.org/10.1073/pnas.1613499114

Rößling, A.-K. et al. eLife 13, RP96943 (2024): https://doi.org/10.7554/eLife.96943.3

Rößling, A. et al. BMC Methods (2026): https://doi.org/10.1186/s44330-026-00063-w

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–3.

Supplementary Software (download ZIP )

Ratiometric image conversion script in Jython (Python) for Fiji/imag J.

Supplementary Data (download XLSX )

Raw data for Supplementary Fig. 1a,d.

Source data

Source Data Fig. 4 (download XLSX )

Numerical source data for Fig. 4 anticipated results.

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Rößling, AK., Mayle, N., Guerard, L. et al. Quantitative imaging of apoplastic pH in plant roots via confocal microscopy. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01337-x

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