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. 2019 Aug 21;9:12188. doi: 10.1038/s41598-019-48455-z

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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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Implementation of circle scanning multiangle TIRF (MA-TIRF). (a) Concept of MA-TIRF. Top: The evanescent excitation field intensity (solid lines) as a function of height above the glass-liquid interface and penetration depth (dashed lines) for various angles of incidence. Lower left: Schematic representation of fluorescent probes (red dots) at 300, 200 and 100 nm (3, 2 and 1, respectively) from the glass-liquid interface. Lower right: Theoretical intensity profiles as a function of angle of incidence for the 3 fluorescent probes. (b) Widefield and height reconstructions of phalloidin-AF560 and mEmerald-Zyxin in a fixed HeLa cell. Scale bars 25 μm. (c) Enlarged view of the leading edge of the cell in b. Scale bars 5 μm. (d) Reconstructed actin height profile (solid black line, red box in c) and zyxin height distribution (green bars, non-zero fits from c).