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. 2019 Dec 23;9:19615. doi: 10.1038/s41598-019-55804-5

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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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In vivo FLIM-FRET analysis in MSR-1 cells with MamJ-phiYFP and MamJ-phiYFP + TagRFP657-MamK. (a) Representative phiYFP (donor) fluorescence decay curves measured by time-correlated single-photon counting (TCSPC) in MSR-1 expressing either MamJ-phiYFP (yellow data points) or MamJ-phiYFP together with TagRFP657-MamK (green data points) translational fusions. For MamJ-phiYFP alone, the data was fitted to a single-exponential deconvolution fitting model (orange line), yielding the decay time τ_D = 2.95 ns. For MamJ-phiYFP in the presence of TagRFP657-MamK, the data was fitted to a bi-exponential deconvolution fitting model (green line), resulting in τ_D = 2.94 ns (fixed value, determined from n = 9, SI) and τ_DA = 0.56 ns. (b) FLIM images of MSR-1 with MamJ-phiYFP alone or (c) together with TagRFP657-MamK. The amplitude-weighted average decay time for phiYFP is shown in pseudo-color code, in which warmer colors represent the unaffected donor decay time and colder colors represent shorter decay times due to energy transfer between phiYFP and TagRFP657. Scale bar: 1 µm.