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. 2017 Sep 18;8:577. doi: 10.1038/s41467-017-00606-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — SRpHi probes respond to pH changes in vivo. a MEF cells pulsed with SRpHi1 for 10 min were equilibrated to the indicated pH by application of an external citric acid/phosphate buffer supplemented with 140 mM KCl and 1 µM nigericin. Cells were imaged in confocal mode. EYFP images have been normalized to the corresponding STAR410 image for visualization. Scale bar: 5 µm. b Cells were imaged as in a with STED. A mask of all endosomes was created and the ratio of EYFP/STAR410 signal was calculated for each endosome (n > 150 endosomes for each pH value), averaged for each pH value, and their log₁₀ value plotted. Error bars represent standard deviation from the mean. Log₁₀ EYFP/STAR410 values were fit to a linear equation (R ² = 0.99)