Nuclear pores as versatile reference standards for quantitative superresolution microscopy.
Journal
Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604
Informations de publication
Date de publication:
10 2019
10 2019
Historique:
received:
19
03
2019
accepted:
14
08
2019
entrez:
29
9
2019
pubmed:
29
9
2019
medline:
15
2
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31562488
doi: 10.1038/s41592-019-0574-9
pii: 10.1038/s41592-019-0574-9
pmc: PMC6768092
mid: NIHMS1537548
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1045-1053Subventions
Organisme : NIDA NIH HHS
ID : U01 DA047728
Pays : United States
Organisme : NIBIB NIH HHS
ID : U01 EB021223
Pays : United States
Organisme : Medical Research Council
ID : MR/K015826/1
Pays : United Kingdom
Organisme : European Research Council
ID : 724489
Pays : International
Organisme : Wellcome Trust
ID : 203276
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M022374/1
Pays : United Kingdom
Commentaires et corrections
Type : ErratumIn
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