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. 2020 Mar 20;10:5146. doi: 10.1038/s41598-020-61478-1

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© The Author(s) 2020

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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Fluorescence lifetime imaging of HeLa cells expressing an mTurq2-Epac1-td^dVenus biosensor. (a) Representative fluorescence intensity, corresponding phasor plots, average fluorescence lifetime FLIM images, and images of the fractional contribution of the open “activated” biosensor of a HeLa cell expressing mTurq2-Epac1-td^dVenus biosensor at 4 time points following stimulation with forskolin (25 µM) and IBMX (100 µM). Images are 232 × 232 pixels with a field of view of 40 µm × 40 µm, an acquisition time of 2 s per frame and pixel binning of 7 × 7 pixels. (b) A representative phasor plot showing the cluster of data points prior to stimulation and the time-dependent position of the data cluster after stimulation (open black circles). The trajectory of the phasor as a function of time after stimulation can be well fitted to a straight line (solid red line). The phasor positions from control measurements from HeLa cells expressing empty vector mTurq2 (open green circles) lie on or very close to the universal circle. Three fluorescence lifetime contributions calculated from multi-image global fits to time-lapse imaging data (solid green circles) are shown along with reference calculated positions for several monoexponential fluorescence lifetimes (filled magenta circles). (c) Plots of u, v from the phasor plots, and total intensity values from the time-lapse measurements.