scholarly journals Nanoscale organization of rotavirus replication machineries

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yasel Garcés Suárez ◽  
Jose L Martínez ◽  
David Torres Hernández ◽  
Haydee Olinca Hernández ◽  
Arianna Pérez-Delgado ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Spatial Resolution ◽  
Structural Organization ◽  
Super Resolution ◽  
Viral Proteins ◽  
Genome Replication ◽  
Molecular Scale ◽  
Viral Components ◽  
Super Resolution Microscopy ◽  
Outermost Zone

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

scholarly journals Nanoscale organization of rotavirus replication machineries

2018 ◽  
Author(s):  
Yasel Garcés ◽  
José L. Martínez ◽  
David T. Hernández ◽  
Haydee O. Hernández ◽  
Mayra Méndez ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Structural Organization ◽  
Super Resolution ◽  
Viral Proteins ◽  
Genome Replication ◽  
Viral Dsrna ◽  
Molecular Scale ◽  
Viral Components ◽  
Super Resolution Microscopy ◽  
Outermost Zone

AbstractRotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viro-plasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins and viral dsRNA within and around the VPs. The observed viral components are spatially organized as 6 concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6 proteins and the dsRNA. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

scholarly journals Structured illumination microscopy and its new developments

2016 ◽  
Vol 09 (03) ◽  
pp. 1630010 ◽  
Author(s):  
Jianling Chen ◽  
Caimin Qiu ◽  
Minghai You ◽  
Xiaogang Chen ◽  
Hongqin Yang ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Spatial Resolution ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Living Cells ◽  
The Other ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Recent Developments ◽  
Super Resolution Microscopy

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

scholarly journals MINSTED fluorescence localization and nanoscopy

2021 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

scholarly journals Super-resolution microscopy and its applications in neuroscience

2017 ◽  
Vol 10 (05) ◽  
pp. 1730001 ◽  
Author(s):  
Xuecen Wang ◽  
Jiahao Wang ◽  
Xinpei Zhu ◽  
Yao Zheng ◽  
Ke Si ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Biological Science ◽  
Basic Principles ◽  
Suitable Tool ◽  
Subcellular Processes ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Optical microscopy promises researchers to see most tiny substances directly. However, the resolution of conventional microscopy is restricted by the diffraction limit. This makes it a challenge to observe subcellular processes happened in nanoscale. The development of super-resolution microscopy provides a solution to this challenge. Here, we briefly review several commonly used super-resolution techniques, explicating their basic principles and applications in biological science, especially in neuroscience. In addition, characteristics and limitations of each technique are compared to provide a guidance for biologists to choose the most suitable tool.

scholarly journals Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy

2020 ◽  
Author(s):  
Milan Vala ◽  
Łukasz Bujak ◽  
Antonio García Marín ◽  
Kristýna Holanová ◽  
Verena Henrichs ◽  
...  
Keyword(s):  
Optical Microscopy ◽  
Electron Micrographs ◽  
Structural Changes ◽  
Structural Transition ◽  
Super Resolution ◽  
Label Free ◽  
Discrete Process ◽  
Static Electron ◽  
Structural Fluctuations ◽  
Super Resolution Microscopy

AbstractMicrotubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, a label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with enrichment of ordered protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a mechanism of switching from the disassembly to the assembly phase.

Resolution in near-field optical microscopy

1991 ◽  
Vol 49 ◽  
pp. 454-455
Author(s):  
M. Isaacson
Keyword(s):  
Optical Microscopy ◽  
Near Field ◽  
Optical Resolution ◽  
Super Resolution ◽  
Optical Probe ◽  
Basic Principles ◽  
Scanning Optical Microscopy ◽  
Super Resolution Microscopy ◽  
Near Field Scanning ◽  
Probe Source

It has only been within the last half decade that the concept of super resolution microscopy in the near-field has been vigorously pursued and experimentally demonstrated. However, the idea of optical resolution unhindered by far field diffraction limitations was conceived more than a half century ago by Synge and further elaborated by O'Keefe in the fifties. That die method was possible, however, was only first demonstrated using 3cm wavelength microwaves almost 20 years later.The basic principles of the method of near field scanning optical microscopy (NSOM) have been described before in the literature. Briefly, the idea is as follows: if an optical probe (source or detector) of diameter D is positioned within a distance of approximately D/π from the surface of an object, and the reflected, transmitted or emitted light is detected, then the lateral spatial region from which the information occurs is limited to aregion of approximate size D and not by the wavelength of the illuminated or detected light.

scholarly journals Spatial Resolution of Coherent Cathodoluminescence Super-Resolution Microscopy

ACS Photonics ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 1067-1072 ◽  
Author(s):  
Joris Schefold ◽  
Sophie Meuret ◽  
Nick Schilder ◽  
Toon Coenen ◽  
Harshal Agrawal ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals MINSTED fluorescence localization and nanoscopy

2020 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

scholarly journals Phosphorylated paxillin and FAK constitute subregions within focal adhesions

2020 ◽  
Author(s):  
Michael Bachmann ◽  
Artiom Skripka ◽  
Bernhard Wehrle-Haller ◽  
Martin Bastmeyer
Keyword(s):  
Focal Adhesion ◽  
Structural Organization ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Multiple Signaling

AbstractIntegrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. We used structured illumination microscopy (SIM) to analyze spatial organization of paxillin phosphorylation (pPax) within adhesions. We found that pPax and focal adhesion kinase (FAK) form spot-like, spatially defined clusters within adhesions in several cell lines. In contrast, other adhesion proteins showed no consistent organization in such clusters. Live-cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify distance to each other dynamically. Moreover, we show that the distance between separate clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural organization within focal adhesions and demonstrate its regulation and dynamics.

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