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. 2017 Nov 15;7:15609. doi: 10.1038/s41598-017-15411-8

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© The Author(s) 2017

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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SensorFRET analysis of MDCK cells expressing TV40 unimolecular FRET constructs incorporated into E-cadherin transmembrane proteins (denoted TS). (A–E) show analysis for the raw data while (F–J) show analysis results for the same data after a 5 pixel Gaussian blur. Panels A/F shows a false color intensity image (by overlaying the Teal and Venus peak intensities), panels B/G show the FRET efficiency maps calculated on a per pixel basis, panels C/H show the FRET efficiency standard deviation (estimated from Supplemental Figure S7, using the measured FRET efficiency and Normalized Fit Residual). Panels D/I show the histogram of measured FRET efficiency values taken from B/G on the primary axis along with the unloaded TL control (image not shown), while the secondary axis shows the estimated Force (in pN) as a function of FRET efficiency. The intersection of the median FRET Efficiency for the tension sensor and the FRET vs Force plot implies the median force per molecule is approximately 1.7 pN. Panels E/J show the estimated force mapped on a per pixel basis, with the inset boxplots showing that differences can only be discerned between regions within a single image once the image is blurred.