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. 2018 Apr 26;8:6610. doi: 10.1038/s41598-018-24899-7

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© The Author(s) 2018

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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Physiological state of [Ca²⁺]_i in the parakeratotic epidermis and mucous epithelia. Representative two-photon images of GCaMP3 mice (a,b) or GCaMP3/tdTomato (c), in a vertical view. (a) In vivo observation of the tail skin. (b,c) Ex vivo observation of the mucous epithelia of the (b) vagina and (c) oral cavity. The contrast in the fluorescence of GCaMP3 was corrected using the fluorescence signal of tdTomato, which reflects the concentration of fluorescent proteins in the cytoplasm. Arrowheads: cells with elevated fluorescence of GCaMP3 beneath the cornified layer. Dashed lines: dermo-epidermal junction or lower edge of the mucous epithelia. Green: GCaMP3; red: autofluorescence (a); blue: second-harmonic generation from dermal collagen. Double-headed arrows: epithelial layers. Scale bars: 50 μm. Results are representative of at least three independent experiments.