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. 2021 Jan 22;12:517. doi: 10.1038/s41467-020-20863-0

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© The Author(s) 2021

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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Fig. 3 — a Microtubules in COS-7 cells. Left: top view. Right: magnified side-views along the numbered lines in the top view, together with the axial profile of the boxed areas. Top- and side-views have the same z-colour scale. Three independent experiments were performed with similar results. b Distribution of microtubule diameters (n = 50), with an average of 41 nm and a standard deviation of 6 nm. An average (n = 8) microtubule profile is also shown. c Histograms of $σ_{\hat{z}}$ at different z-positions, obtained experimentally from 366 DNA-PAINT single-molecule traces. Data include traces from microtubules in COS-7 cells, as well as from spectrin in hippocampal neurons (Fig. 2d). d Histogram of the experimentally determined variance of $\hat{N}$ ( $σ_{\hat{N}}$ ), expressed in units of $\sqrt{\hat{N}}$ to remark how much larger is $σ_{\hat{N}}$ with respect to the expected theoretical lower bound ( $σ_{\hat{N}} = \sqrt{\hat{N}}$ ). The values 2, 5 and 8 are highlighted. The variance was obtained from the same 366 single-molecule traces as those analysed in c. e Median $σ_{\hat{z}}$ values located in the median z-position of each interval defined in c, overlapped with theoretical curves of $σ_{\hat{z}}$ for $\hat{N}$ ₀ = 51,000 and $σ_{\hat{N}}$ = 2x, 5x or 8x $\sqrt{\hat{N}}$ for σ_dF = 2 and 4 nm. Error bars represent the standard deviation of $σ_{\hat{z}}$ and z. The number of single-molecule traces analysed in each interval was 93 (for z = 35–90 nm); 90 (for z = 90–115 nm); 87 (for z = 115–150 nm); and 96 (for z = 150–225 nm). Data includes localizations from three biologically independent samples (immunolabeled microtubules in two independent COS-7 cells and immunolabeled spectrin in a hippocampal neuron). Scale bars represent 1 µm (a, top view); 50 nm (a, side view) and 25 nm (b). Number of localizations kept after frame-filtering step: a (1) 176 (out of 292), (2) 257 (out of 433), (3) 131 (out of 294) and (4) 85 (out of 275).