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. 2020 Apr 15;11:1848. doi: 10.1038/s41467-020-15687-x

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© The Author(s) 2020, corrected publication 2024

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. 3 — a Epac1-camps biosensor is utilized to demonstrate the advantage of stagRFP, where the same sensor design yields a viable FRET biosensor using stagRFP, but not using the ultra-bright RFP mScarlet. The change in normalized emission ratio (red/green) upon 50 μM forskolin and 100 μM IBMX stimulation is shown (stagRFP, n = 8 biologically independent cells; mScarlet, n = 11). The mean ratio ± SEM is displayed. b The response to adrenergic stimulation (1 μM isoproterenol) in HEK293T cells is captured by GR-ICUE2 at high dynamic range. The change in normalized emission ratio (green/red) is shown (n = 20). The mean ratio ± SEM is displayed. c The dynamic range of GR-ICUE2 is comparable to, but still significantly smaller (*p = 0.03, t = 2.399, df = 17, from two tailed, unpaired Welch’s t-test) than the best cyan-yellow cAMP biosensor ICUE3; upon Fsk and IBMX stimulation, the kinetics of GR-ICUE2 are not significantly different from those of ICUE3 (ICUE3, n = 12; GR-ICUE2, n = 14). The mean ratio ± SEM is displayed, and n. s. is not significant. Source data for c is provided as a Source Data file.