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. 2020 Jul 17;11:3607. doi: 10.1038/s41467-020-17444-6

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© The Author(s) 2020

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. 2 — a 3D trajectory of a single Atto 647 N molecule tracked for 160 s in 90 wt% glycerol. XYZ coordinates are extracted from the readout of the pieozelectric nanopositioner during active feedback. b Intensity trace recorded during the trajectory shown in (a), showing uniform intensity despite the rapid diffusion of the molecule. c MSD versus lag time and linear fit of the trajectory in (a), yielding a hydrodynamic diameter of 0.98 nm, consistent with a single fluorophore. d Histogram of diffusion coefficients and hydrodynamic diameters of a population of Atto 647 N trajectories (number of trajectories n = 38). e Photon-pair correlation trace for the molecule in Fig. S6a, showing anti-bunching of photon arrivals at short lag time. f, g Comparison of single-molecule data collection with feedback (f) off and (g) on. Histograms demonstrate the increase in photon collection with the feedback enabled, as well as the uniform observed emission rate from tracked fluorophores.