Automated STED nanoscopy for high-throughput imaging of cellular structures

Mol, Frank N.; Vlijm, Rifka

doi:10.1101/2022.09.29.510126

Cited by 3 publications

(2 citation statements)

References 30 publications

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“…For the detection of the 560 channel (561-nm laser excitation), the detection filter was set to 603/27 nm. For image acquisition, automated STED has been used as presented in (49). For image analysis, a script was written in Python 3 to quantify the relative protein ring positions, available at (50).…”

Section: Sted Microscopymentioning

confidence: 99%

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The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

et al. 2023

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ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA–adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III–dependent division system in Sulfolobus acidocaldarius , one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III–dependent membrane remodeling.

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Section: Sted Microscopymentioning

confidence: 99%

“…For image acquisition, automated STED has been used as presented in (49). For image analysis, a script was written in Python 3 to quantify the relative protein ring positions, available at (50).…”

Section: Sted Microscopymentioning

confidence: 99%

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

et al. 2023

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Fluorescence-based super-resolution-microscopy strategies for chromatin studies

Burgers

Vlijm

2023

Chromosoma

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Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To illustrate how SRM-based methods can significantly improve our understanding of chromatin functioning, and to serve as an inspiring starting point for future work, we conclude with examples of recent applications of SRM in chromatin research.

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Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

Berrevoets,

Kessler,

Balakrishnan

et al. 2024

Preprint

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Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super-resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we targeted the 3D organization of an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstructed the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of RNA organization. By integrating multi-positional labeling, we determined the coarse conformation of the RNA within the organelle and found the 3' end forming a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of nuclear paraspeckle structure and growth, as well as on the molecular organization of the scaffold RNA.

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Automated STED nanoscopy for high-throughput imaging of cellular structures

Cited by 3 publications

References 30 publications

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

Fluorescence-based super-resolution-microscopy strategies for chromatin studies

Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

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