Optical nanoscopy

Diaspro, Alberto; Bianchini, Paolo

doi:10.1007/s40766-020-00008-1

Cited by 26 publications

(30 citation statements)

References 177 publications

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“…Confocal and Stimulated Emission Depletion (STED) nanoscopy were performed as previously reported (Diaspro and Bianchini, 2020;Vicidomini et al, 2018). aNPCs were plated on glass coverslips 24 h before fixation.…”

Section: Immunofluorescence Sted Nanoscopy and Particle Analysismentioning

confidence: 99%

The piRNA pathway sustains adult neurogenesis by reducing protein synthesis and cellular senescence

Gasperini

Tuntevski

Pelizzoli

et al. 2020

Preprint

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In specific niches of the adult mammalian brain, neural progenitor cells (aNPCs) ensure lifelong neurogenesis. Proper regulation of this process entails important implications for brain plasticity and health. We report that Piwil2 (Mili) and PIWI-interacting RNAs (piRNAs) are abundantly expressed in aNPCs but depleted in their progeny in the adult mouse hippocampus. Loss of function of the piRNA pathway in aNPCs inhibited neurogenesis and increased reactive gliogenesis in vivo and in vitro. PiRNA pathway depletion in cultured aNPCs increased levels of 5S ribosomal RNA, transfer RNAs and mRNAs encoding regulators of translation, resulting in higher polyribosome density and protein synthesis upon differentiation. We propose that the piRNA pathway sustains adult neurogenesis by repressing translation in aNPCs.One sentence summaryThe piRNA pathway is enriched in neural precursors and essential for appropriate neurogenesis by modulating translation

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Section: Immunofluorescence Sted Nanoscopy and Particle Analysismentioning

confidence: 99%

The piRNA pathway sustains adult neurogenesis by reducing protein synthesis and cellular senescence

Gasperini

Tuntevski

Pelizzoli

et al. 2020

Preprint

Self Cite

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“…It took about a century for establishing that the Abbe’s diffraction limit holds from a physical point of view, but it can be effectively circumvented by adding additional information when forming an image [ 1 , 6 ]. The 2014 Nobel prize in Chemistry awarded three scientists whose seminal work demonstrated “super-resolution” (i.e., below Abbe’s limit) imaging by leveraging new detection modalities which combine localization precision or shaping of the illumination beam with the emission bistability of fluorophores under certain conditions.…”

Section: Introductionmentioning

confidence: 99%

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

Storti

Carlotti

Chiellini

et al. 2022

IJMS

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In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

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“…well below the optical resolution of confocal and TIRF microscope on the focal plane (200-300 nm), and details of single viral particles interacting with subcellular structures may be only partially revealed by these techniques. Optical super-resolution methods that break the light-diffraction barrier either by leveraging on the photophysical properties of the fluorescent probe, or by structuring the excitation light, may easily reach the 20-150 nm spatial scale 32 . Indeed, STimulated Emission Depletion (STED) and Single Molecule Localization Microscopy (SMLM) have been recently applied to image single viruses of different families at<100 nm, also in the cellular context 33,34,35 .…”

Section: Introductionmentioning

confidence: 99%

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

Storti

Quaranta

Primio

et al. 2021

Preprint

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We developed a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy (Airyscan, STORM, and STED) and easily matches the spatial scale of single virus-cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in VeroE6 cells. Our results suggest that the variant of concern B.1.1.7, currently on the rise in several countries by a clear transmission advantage, in these cells outcompetes its ancestor B.1 in terms of a much faster kinetics of entry. Given the molecular scenario (entry by the late pathway and similar fraction of pre-cleaved S protein for B.1.1.7 and B.1), the faster entry of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike RBD with ACE2. Remarkably, we also observed directly the significant role of clathrin as mediator of late entry endocytosis, which had been previously suggested in analogy with other CoVs and from experiments on pseudotyped virus models. On overall, we believe that our fluroescence microscopy-based approach is valuable for future studies addressing of how SARS-CoV-2 and its variants interact with cells.

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Optical nanoscopy

Cited by 26 publications

References 177 publications

The piRNA pathway sustains adult neurogenesis by reducing protein synthesis and cellular senescence

The piRNA pathway sustains adult neurogenesis by reducing protein synthesis and cellular senescence

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

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