Assessment of 3D MINFLUX data for quantitative structural biology in cells
Assessment of 3D MINFLUX data for quantitative structural biology in cells
复制标题
评估细胞定量结构生物学的 3D MINFLUX 数据
DOI：
10.1038/s41592-022-01694-x
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发表时间：
2022
期刊：
影响因子：
48
通讯作者：
Prakash K
中科院分区：
文献类型：
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作者：
Prakash K
In 2017, Balzarotti et al. 1 introduced MINFLUX to localize individual fluorophores in single-molecule localization microscopy by probing the emitter with a donut-shaped excitation beam 1. The authors attained a precision∼ 1 nm and resolved loci on DNA origami placed 6 nm apart. In 2020, Gwosch et al. 2 extended the method to fixed and living biological cells, and into three-dimensional (3D) localization and two colors, claiming resolutions in the range of 1–3 nm for subcellular structures 2. Using nuclear pore complexes (NPCs) as an example, the authors measured localization precisions of 1–3 nm and asserted (1) that MINFLUX can clearly resolve the eightfold symmetry of Nup96 in single nuclear pores;(2) Nup96 is distributed along a ring of 107 nm in diameter; and (3) that 3D MINFLUX can resolve the parallel cytoand nucleoplasmic layers of Nup96 in single pore complexes,~ 50 nm apart in the axial (z) direction. However, we were not convinced by the evidence given for these claims and have therefore reanalyzed the datasets provided by the authors.We agree with their main localization precision results, but in our reanalysis we found (1) that the eightfold symmetry of NPCs is rarely visible at a single nuclear pore level and was not clearly determined in structure-based modeling of the localization datasets;(2) that the mean or best-fit Nup96 ring diameter varies between datasets and the spread of diameters in each dataset is broader than that found by dSTORM 3; and (3) the average z-distance between cyto-and nucleoplasmic layers of Nup96 localizations was 40.5 nm instead of~ 50 nm, in the dataset on which this claim was based. Furthermore, in two-color imaging, the inner ring found in similar dSTORM experiments at 40-nm diameter 3, 4 was not resolved as a ring by MINFLUX. We therefore conclude that while these MINFLUX datasets demonstrate high 3D precision in localizing molecules, they do not appear to demonstrate the accuracy of previously published state-of-the-art dSTORM imaging of NPCs 3.
重新审视细胞定量结构生物学的 3D MINFLUX 数据评估
DOI：
--
发表时间：
2022
期刊：
bioRxiv
影响因子：
--
作者：
K. Gwosch;F. Balzarotti;Jasmin K. Pape;P. Hoess;J. Ellenberg;J. Ries;U. Matti;R. Schmidt;S. Sahl;Stefan W. Hell
通讯作者：
Stefan W. Hell
影响因子：
16.6
作者：
Heydarian H;Joosten M;Przybylski A;Schueder F;Jungmann R;Werkhoven BV;Keller-Findeisen J;Ries J;Stallinga S;Bates M;Rieger B
通讯作者：
Rieger B