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. 2022 Oct 10;41(4):552–559. doi: 10.1038/s41587-022-01489-7

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© The Author(s) 2022

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. 1 — a, Anisotropy values and temporal regime accessible to conventional time-resolved anisotropy and STARSS. By continuously probing the same subset of molecules populating the same reversible switchable state, it is possible to extend the observable time window to microseconds and milliseconds. This previously inaccessible temporal regime enables measurement of slower rotational diffusion. b, STARSS can measure slower rotational diffusion coefficients and thereby quantify the mass and mass changes of large protein complexes, as well as complex dynamics, in a local viscous environment. The relationship between rotational diffusion time and the size of complexes is shown, together with the domain of applicability of techniques. c, Size of proteins and biological complexes that can be investigated with STARSS.