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. 2019 Mar 18;9:4829. doi: 10.1038/s41598-019-40931-w

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© The Author(s) 2019

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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Effect of FRET sensors to [Ca²⁺] dynamics in HeLa cells. (a–d) Fluorescent images of IRIS-2 expressing cells without (a,b) and with HaloTag TMR staining (c,d). Bar, 50 μm. Emissions detected by filters for GFP are shown in (a,c). Emissions detected by filters for TMR are shown in (b,d). (e) A fluorescent image of DYC3.60 expressing cells. (f–h) Effect of FRET sensors to [Ca²⁺] dynamics in HeLa cells. Cells expressing IRIS-2 (f), IRIS-2-Dmut (g), or DYC3.60 (h) were stained with HaloTag TMR and Indo-5F. Left panels are time courses of emission ratio (f,g) or emission (h) changes of FRET sensors in mGluR5 expressing HeLa cells stimulated with 10 μM glutamate (horizontal bars). Right panels are time courses of emission changes of Indo-5F in the same cells of left panels. (i–k) [IP₃] and [Ca²⁺] changes were imaged with the dual FRET probes. Initiation of the first Ca²⁺ spike and the termination of Ca²⁺ oscillations were marked with dashed line with closed and open triangles, respectively. (j) [IP₃] and [Ca²⁺] changes around the rise of first Ca²⁺ spike. (k) A phase plane trajectory is drawn with [IP₃] and [Ca²⁺] imaging data.