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. 2018 Jan 30;8:1866. doi: 10.1038/s41598-018-20251-1

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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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Increased sensitivity of R-FlincA. (a) Experimental design for dual-colour cAMP imaging in combination with extra-cellular uncaging of Bhc-cAMP³⁷. (b) Representative signal changes for R-FlincA and Flamindo2 in a single D. discoideum cell. Arrows indicate the timing of uncaging (405 nm, 6.7 μsec) with increasing power. (c) Signal changes of R-FlincA and Flamindo2 (absolute values of ΔF/F₀) in response to extra-cellularly supplied cAMP with increasing uncaging power. Bars represent +SEM, N = 10 cells for laser power 0, 0.1, and 0.25%; N = 5 for 0.5%; N = 15 for 2.5%. Asterisks represent significant differences compared to the control group (0%) at p < 0.05 (Student’s t test with two-tailed). (d) Spontaneous cAMP dynamics in the population of D. discoideum cells, detected by the ratiometry of R-FlincA and Flamindo2. The small pulse amplitude at early development (magenta circles) is detected with R-FlincA, but not with Flamindo2. Black circles are the large cAMP pulses detected by both R-FlincA and Flamindo2. F₀ and R₀ is the fluorescence intensity and ratio value at 6 h, respectively. Ratio values were obtained from the pixel-by-pixel calculation. The averaged data in 0.08 mm² containing ~100 cells.