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. 2019 Oct 2;9:14219. doi: 10.1038/s41598-019-50733-9

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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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Knock-in of codon optimised mEos 4b in Hek293T results in a 2-fold improvement in expression compared to mEos 3.2. (A) mEos 4b CO knock-in Hek293T were generated and compared to previously reported mEGFP and mEos 3.2 knock-ins. A 2-fold, significant improvement in expression is observed in western blotting when compared to mEos 3.2 (*p = 0.0473, ns p = 0.49, *p = 0.0444 clones D2, D4, and E2 respectively). (B) mEos 4b CO labelled cells demonstrate improved expression of tagged TUBA1B, when compared to mEos 3.2 knock-in clones observe as an increase in (C) the number of localisations (**p = 0.0053), (D) FRC (**p = 0.0010), and (E) modal XY uncertainty (*p = 0.0210) when compared to mEos 3.2. (n = 3 (S.D), Western Blotting tested by 2-way ANOVA with multiple comparisons, SMLM quantifications by t-testing. Complete unedited gels are included in Fig. S6) FRC mreasurements were taken from an ROI cropped from the centre of each image, number of localisations and XY uncertainty are measured from the whole filed of view.