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. 2017 Sep 12;7:11379. doi: 10.1038/s41598-017-11563-9

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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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Qdot labelling fails to distinguish substrate-dependent changes in receptor diffusion. (A) Schematic representation of coverslips functionalized with (i) poly-L-lysine, (ii) anti-MHCII antibodies (αMHCII), or (iii) fibronectin, indicating the mode of cell attachment and the assumed differences in the distance between the plasma membrane and the coverslip. (B) Ex vivo murine splenic B cells were labelled and seeded onto the indicated functionalized coverslips (upper panel poly-L-lysine, middle panel αMHCII, lower panel fibronectin) before imaging by TIRFM. Trajectories are plotted using a colour-coded temporal scale. The dashed lines indicate cell boundaries. Scale bars = 5 μm. (C,D) Single-state diffusion coefficients for IgM-BCR tracks obtained using Fab-Cy3 labelling (C) or Fab-biotin-Qdot labelling (D). For all three substrates, the cumulative frequency curves of the diffusion coefficients are shown and the median values are indicated by the dots on the curves.