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Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Thevathasan, JV; Kahnwald, M; Cieslinski, K; Hoess, P; Peneti, SK; Reitberger, M; Heid, D; ... Ries, J; + view all (2019) Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nature Methods , 16 pp. 1045-1053. 10.1038/s41592-019-0574-9.

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Abstract

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

Type: Article
Title: Nuclear pores as versatile reference standards for quantitative superresolution microscopy
DOI: 10.1038/s41592-019-0574-9
Publisher version: https://doi.org/10.1038/s41592-019-0574-9
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10084914
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