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. 2017 Jun 9;8:15732. doi: 10.1038/ncomms15732

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Copyright © 2017, The Author(s)

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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(a) Two-colour time-lapse TIRFM images of Fluo4 (green) and Alexa Fluor 647-Fab anti-TCR α/β (red) captured at the indicated time points (Supplementary Movie 1) from isolated Tespa1^+/+, Tespa1^−/− and Tespa1-FFAA (FFAA) DP cells placed on planar lipid bilayers containing anti-CD3 antibodies. See Supplementary Movie 1 for the complete time-lapse TIRFM images. Scale bar, 3 μm. (b) Normalized fluorescence intensity (FI) of Ca²⁺ imaging (Fluo4) from high-speed, high-resolution, time-lapse TIRFM images of real-time Ca²⁺ flux in the TCR-proximal region in isolated Tespa1^+/+ (+/+), Tespa1^−/− (−/−) and Tespa1-FFAA (FFAA) DP cells as in a. Data are representative of at least three experiments. Bars represent mean±s.e.m. of 10 (FFAA) or 12 (+/+ and −/−) cells from one representative of three independent experiments.