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. 2017 Aug 1;7:6994. doi: 10.1038/s41598-017-06960-z

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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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Schematic representation of moving subtrajectory analysis and kinetics analysis. (A) Shown is a gallery of single-molecule trajectories of Qdot 585-labeled CD45 (cyan) and Qdot 655-labeled CD3ε (red) superimposed upon 200-frame averaged CD3ζ-EGFP images, which were obtained by analysis of simultaneous triple-color single-molecule movies recorded at 33.33 ms/frame. Arrows indicate trajectories inside TCR microclusters. Bar, 1 μm. (B) Standard MSD analysis using single-molecule tracking. (C) Moving subtrajectory MSD analysis. All subtrajectories were composed of successive N _sub spots (N _sub = 11 in the present study), i.e., (N _sub − 1) steps were extracted from each trajectory, and their MSD curves were calculated. (D–F) Representative examples of sorting of the subtrajectories into the three diffusion types: simple (D), directional (E), and confined (F). After each subtrajectory was fitted to all three equations for the simple (Equation 5, green), directional (Equation 6, blue), and confined (Equation 7, red) diffusion, it was assigned to the diffusion type giving the least residual standard error of the fitting (solid line: assigned, dashed line: not assigned). Within 10 data points of the MSD of a single subtrajectory, the first 5 points (filled circle) were used for the fitting and the later points (open circle) were not. (G) Schematic representation of sorting of the subtrajectories of a trajectory into the three location groups: the inside (red), boundary (blue), and outside (green) of the microcluster.