Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging

Ong, Wei Qiang; Citron, Y. Rose; Schnitzbauer, Joerg; Kamiyama, Daichi

doi:10.1039/c5cc04575d

Cited by 27 publications

(14 citation statements)

References 31 publications

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“…Our approach has focused mainly on creating the sharpest PSF and detecting as much of the emitted fluorescence light as possible. Recently, there have been promising developments that increase the number of emitted photons per molecule ( Klehs et al., 2014 , Ong et al., 2015 , Vaughan et al., 2012 ), which we have not exploited here. Unfortunately, these advances have so far come at the expense of an increase in recording time.…”

Section: Discussionmentioning

confidence: 99%

Ultra-High Resolution 3D Imaging of Whole Cells

Huang

Sirinakis

Allgeyer

et al. 2016

Cell

278

292

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SummaryFluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

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Section: Discussionmentioning

confidence: 99%

Ultra-High Resolution 3D Imaging of Whole Cells

Huang

Sirinakis

Allgeyer

et al. 2016

Cell

278

292

View full text Add to dashboard Cite

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“…Although much is known about externally modifying the photophysical properties of organic dye molecules for single-molecule imaging 30 , 31 , 48 , 49 , there are currently few ways of altering those of PM FPs, almost certainly due to the relative inaccessibility of the chromophore in the interior of the β-barrel of the protein 50 , 51 . Since most PM fluorophores are currently still fluorescent proteins, our approach offers a way to positively modify them while preserving their specific properties of photo-activation/conversion.…”

Section: Discussionmentioning

confidence: 99%

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells

et al. 2018

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A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

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“…For single-color superresolution imaging, samples were mounted in a D 2 O-based imaging buffer (50 mM Tris-HCl pH 8 in 95% D 2 O) to improve brightness ( Ong et al., 2015 ). After selection of a region of interest, the back focal plane was imaged to ensure that the immersion oil contained no air bubbles.…”

Section: Methodsmentioning

confidence: 99%

Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation

Mund

Beek

Deschamps

et al. 2018

Cell

198

239

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SummaryClathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

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Heavy water: a simple solution to increasing the brightness of fluorescent proteins in super-resolution imaging

Cited by 27 publications

References 31 publications

Ultra-High Resolution 3D Imaging of Whole Cells

Ultra-High Resolution 3D Imaging of Whole Cells

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells

Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation

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