Publisher Correction: Nuclear pores as versatile reference standards for quantitative superresolution microscopy

AbstractQuantitative 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 or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.These cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

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How good are my data? Reference standards in superresolution microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e19-04-0189 ◽

2020 ◽

Vol 31 (19) ◽

pp. 2093-2096

Author(s):

Markus Mund ◽

Jonas Ries

Keyword(s):

Imaging Technique ◽

Reference Standards ◽

Reference Standard ◽

Yield Data ◽

Superresolution Microscopy ◽

Experimental Parameters ◽

Biological Discovery ◽

Microscopy Data ◽

Suitable Reference ◽

Over Time

Superresolution microscopy is becoming increasingly widespread in biological labs. While it holds enormous potential for biological discovery, it is a complex imaging technique that requires thorough optimization of various experimental parameters to yield data of the highest quality. Unfortunately, it remains challenging even for seasoned users to judge from the acquired images alone whether their superresolution microscopy pipeline is performing at its optimum, or if the image quality could be improved. Here, we describe how superresolution microscopists can objectively characterize their imaging pipeline using suitable reference standards, which are stereotypic so that the same structure can be imaged everywhere, every time, on every microscope. Quantitative analysis of reference standard images helps characterizing the performance of one’s own microscopes over time, allows objective benchmarking of newly developed microscopy and labeling techniques, and finally increases comparability of superresolution microscopy data between labs.

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Nuclear Pores as Universal Reference Standards for Quantitative Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2018.11.757 ◽

2019 ◽

Vol 116 (3) ◽

pp. 137a ◽

Cited By ~ 2

Author(s):

Jervis V. Thevathasan ◽

Ulf Matti ◽

Maurice Kahnwald ◽

Sudheer Kumar Peneti ◽

Bianca Nijmeijer ◽

...

Keyword(s):

Reference Standards ◽

Quantitative Microscopy ◽

Nuclear Pores ◽

Universal Reference

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The Formation, Structure, Distribution and Frequency of Nuclear Pores

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066644 ◽

1971 ◽

Vol 29 ◽

pp. 518-519

Author(s):

G. G. Maul

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Membrane ◽

Dynamic Structure ◽

Nuclear Pore ◽

Nuclear Pores ◽

Filter Function ◽

Etching Technique ◽

Selective Filter ◽

Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

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High-voltage cytochemistry for three-dimensional observation of nuclei and calculation of total numbers of nuclear pores in retinal Mueller glia

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016234x ◽

1990 ◽

Vol 48 (3) ◽

pp. 956-957

Author(s):

H. Ishigooka ◽

S. Ueno ◽

L.M. Hjelmeland ◽

M.B. Landers ◽

K. Ogawa

Keyword(s):

Nuclear Envelope ◽

High Voltage ◽

Three Dimensional ◽

Reaction Medium ◽

Nuclear Pores ◽

High Voltage Electron Microscopy ◽

Voltage Electron ◽

High Voltage Electron ◽

G6pase Activity ◽

Mueller Glia

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).

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HGM Reference Standards and Wetland Restoration

Engineering Approaches to Ecosystem Restoration ◽

10.1061/40382(1998)182 ◽

1998 ◽

Author(s):

D. Eric Somerville ◽

Wade L. Nutter

Keyword(s):

Wetland Restoration ◽

Reference Standards

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Regarding the problems of reference standards using

Ferrous Metallurgy Bulletin of Scientific Technical and Economic Information ◽

10.32339/0135-5910-2019-10-1169-1179 ◽

2020 ◽

Vol 75 (10) ◽

pp. 1169-1179

Author(s):

E. P. Dryagun ◽

M. A. Polyakova ◽

О. A. Belan ◽

N. T. Alsynbaev

Keyword(s):

Reference Standards

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The method of determining the metrological performance of unit reference standards of reflection coefficient in waveguides at millimeter waves

Izmeritel`naya Tekhnika ◽

10.32446/0368-1025it.2020-1-59-63 ◽

2020 ◽

pp. 59-63

Author(s):

A.S. Bondarenko ◽

A.S. Borovkov ◽

I.M. Malay ◽

V.A. Semyonov

Keyword(s):

Reflection Coefficient ◽

Millimeter Waves ◽

Primary Standard ◽

Reference Standards ◽

Measurement Scale ◽

Current State ◽

Coefficient Measurement ◽

Mathematical Equations ◽

Electrodynamic Modeling ◽

Metrological Performance

The analysis of the current state of the reflection coefficient measurements in waveguides at millimeter waves is carried out. An approach for solving the problem of reproducing the reflection coefficient measurement scale is proposed. Mathematical equations, which are the basis of the reflection coefficient measurement equation are obtained. The method of determining the metrological performance of reflection coefficient unit’s reference standards is developed. The results of electrodynamic modeling and analytical calculations by the developed method are compared. It is shown that this method can be used for reproducing the reflection coefficient unit in the development of the State primary standard.

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