Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 350-359
Author(s):  
Nobuhiro Morone ◽  
Eiji Usukura ◽  
Akihiro Narita ◽  
Jiro Usukura
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Apical Cell ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy ◽  
Cytoplasmic Surface ◽  
A Cell ◽  
Freeze Etching

Abstract Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

scholarly journals Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Álvaro Ortega-Esteban ◽  
Carlos P. Mata ◽  
María J. Rodríguez-Espinosa ◽  
Daniel Luque ◽  
Nerea Irigoyen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Nucleic Acids ◽  
Capsid Protein ◽  
Cycle Phase ◽  
Human Virus ◽  
Double Stranded Rna ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy

ABSTRACT Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo. IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

scholarly journals Structural identification of individual helical amyloid filaments by integration of cryo-electron microscopy-derived maps in comparative morphometric atomic force microscopy image analysis

2021 ◽  
Author(s):  
Liisa Lutter ◽  
Youssra Al-Hilaly ◽  
Christopher J. Serpell ◽  
Mick F. Tuite ◽  
Claude M. Wischik ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Image Analysis ◽  
Structural Analysis ◽  
Amyloid Fibrils ◽  
Structural Identification ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297-391), termed 'dGAE'. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are closely related structurally to the paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.

Analysis of the Structure of Bacterial Photosynthetic Complexes, and their Arrangement within the Bacterial Photosynthetic Membrane, by Cryo-electron Microscopy and Atomic Force Microscopy

2006 ◽  
Vol 12 (S02) ◽  
pp. 36-37 ◽  
Author(s):  
CN Hunter
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Photosynthetic Membrane ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy ◽  
Photosynthetic Complexes

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

Extracting elastic properties and prestress of a cell using atomic force microscopy

2009 ◽  
Vol 24 (3) ◽  
pp. 1167-1171 ◽  
Author(s):  
C.Y. Zhang ◽  
Y.W. Zhang
Keyword(s):  
Mechanical Properties ◽  
Atomic Force Microscopy ◽  
Elastic Modulus ◽  
Analytical Solution ◽  
Cell Membrane ◽  
Soft Phase ◽  
Force Microscopy ◽  
Apparent Modulus ◽  
Atomic Force ◽  
A Cell

An analytical solution was derived for the indentation of a cell using atomic force microscopy. It was found that the contribution of the cell membrane to the overall indentation stiffness is dependent on the size of the indenter. When a small indenter [for example, an atomic force microscopy (AFM) tip] is used to probe the mechanical properties of cells, the cell membrane and its prestress were important in interpreting indentation data. The solution allows the partition of contributions from the membrane and the interior soft phase. The apparent elastic modulus of the cell and the prestress of the cell membrane can be extracted. In addition, the modulus of the cell membrane could be estimated from the extracted apparent modulus if the interior soft phase of the cell was known and vice versa. However, when a large indenter is used (for example, a microbead attached to the cantilever beam of the AFM), the contribution of the cell membrane is negligible.

scholarly journals Corrosion Behavior and Mechanical Properties of a Nanocomposite Superhydrophobic Coating

Coatings ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 652
Author(s):  
Divine Sebastian ◽  
Chun-Wei Yao ◽  
Lutfun Nipa ◽  
Ian Lian ◽  
Gary Twu
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Scanning Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Nanocomposite Coating ◽  
Superhydrophobic Coating ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Images ◽  
Scanning Electron

In this work, a mechanically durable anticorrosion superhydrophobic coating is developed using a nanocomposite coating solution composed of silica nanoparticles and epoxy resin. The nanocomposite coating developed was tested for its superhydrophobic behavior using goniometry; surface morphology using scanning electron microscopy and atomic force microscopy; elemental composition using energy dispersive X-ray spectroscopy; corrosion resistance using atomic force microscopy; and potentiodynamic polarization measurements. The nanocomposite coating possesses hierarchical micro/nanostructures, according to the scanning electron microscopy images, and the presence of such structures was further confirmed by the atomic force microscopy images. The developed nanocomposite coating was found to be highly superhydrophobic as well as corrosion resistant, according to the results from static contact angle measurement and potentiodynamic polarization measurement, respectively. The abrasion resistance and mechanical durability of the nanocomposite coating were studied by abrasion tests, and the mechanical properties such as reduced modulus and Berkovich hardness were evaluated with the aid of nanoindentation tests.

scholarly journals Cell penetration efficiency analysis of different atomic force microscopy nanoneedles into living cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marcos Penedo ◽  
Tetsuya Shirokawa ◽  
Mohammad Shahidul Alam ◽  
Keisuke Miyazawa ◽  
Takehiko Ichikawa ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Cell Biology ◽  
Cell Damage ◽  
Efficiency Analysis ◽  
Living Cells ◽  
Biomolecular Recognition ◽  
Cell Penetration ◽  
Force Microscopy ◽  
Atomic Force

AbstractOver the last decade, nanoneedle-based systems have demonstrated to be extremely useful in cell biology. They can be used as nanotools for drug delivery, biosensing or biomolecular recognition inside cells; or they can be employed to select and sort in parallel a large number of living cells. When using these nanoprobes, the most important requirement is to minimize the cell damage, reducing the forces and indentation lengths needed to penetrate the cell membrane. This is normally achieved by reducing the diameter of the nanoneedles. However, several studies have shown that nanoneedles with a flat tip display lower penetration forces and indentation lengths. In this work, we have tested different nanoneedle shapes and diameters to reduce the force and the indentation length needed to penetrate the cell membrane, demonstrating that ultra-thin and sharp nanoprobes can further reduce them, consequently minimizing the cell damage.

Characterization of self-assembled Ge islands on Si(100) by atomic force microscopy and transmission electron microscopy

1998 ◽  
Vol 321 (1-2) ◽  
pp. 86-91 ◽  
Author(s):  
G Wöhl ◽  
C Schöllhorn ◽  
O.G Schmidt ◽  
K Brunner ◽  
K Eberl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transmission Electron ◽  
Self Assembled

Study of ageing of ketone resins used as picture varnishes by FTIR spectroscopy, UV–Vis spectrophotometry, atomic force microscopy and scanning electron microscopy X-ray microanalysis

2008 ◽  
Vol 391 (4) ◽  
pp. 1351-1359 ◽  
Author(s):  
María Teresa Doménech-Carbó ◽  
Dolores Julia Yusá-Marco ◽  
Giovanna Bitossi ◽  
Miguel F. Silva ◽  
Xavier Mas-Barberá ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Ftir Spectroscopy ◽  
X Ray ◽  
Force Microscopy ◽  
Atomic Force ◽  
Scanning Electron

scholarly journals Self-lubricant behaviour of copper-carbon nanocomposites: An electron microscopy and atomic force microscopy study

2015 ◽  
Vol 21 (S6) ◽  
pp. 114-115
Author(s):  
D. Silva ◽  
J. Jeremias ◽  
D. Nunes ◽  
J.B. Correia ◽  
K. Hanada ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Microscopy Study ◽  
Atomic Force Microscopy Study ◽  
Carbon Nanocomposites ◽  
Force Microscopy ◽  
Atomic Force
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