scholarly journals Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

2016 ◽  
Author(s):  
Bong-Gyoon Han ◽  
Zoe Watson ◽  
Hannah Kang ◽  
Arto Pulk ◽  
Kenneth H. Downing ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Shelf Life ◽  
Single Crystals ◽  
Convenient Method ◽  
Test Specimen ◽  
Biological Macromolecules ◽  
Crystalline Order ◽  
Binding Function ◽  
Biotin Binding

ABSTRACTWe describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

High-Resolution Cryo-Electron Microscopy of Biological Macromolecules

1990 ◽  
Vol 48 (1) ◽  
pp. 126-127
Author(s):  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Diffraction Pattern ◽  
Superfluid Helium ◽  
Copper Phthalocyanine ◽  
Optical Diffraction ◽  
Irradiation Damage ◽  
Biological Macromolecules ◽  
Cross Sectional ◽  
Instrumental Resolution

The resolution of direct images of biological macromolecules is normally restricted to far less than 0.3 nm. This is not due instrumental resolution, but irradiation damage. The damage to biological macromolecules may expect to be reduced when they are cooled to a very low temperature. We started to develop a new cryo-stage for a high resolution electron microscopy in 1983, and successfully constructed a superfluid helium stage for a 400 kV microscope by 1986, whereby chlorinated copper-phthalocyanine could be photographed to a resolution of 0.26 nm at a stage temperature of 1.5 K. We are continuing to develop the cryo-microscope and have developed a cryo-microscope equipped with a superfluid helium stage and new cryo-transfer device.The New cryo-microscope achieves not only improved resolution but also increased operational ease. The construction of the new super-fluid helium stage is shown in Fig. 1, where the cross sectional structure is shown parallel to an electron beam path. The capacities of LN2 tank, LHe tank and the pot are 1400 ml, 1200 ml and 3 ml, respectively. Their surfaces are placed with gold to minimize thermal radiation. Consumption rates of liquid nitrogen and liquid helium are 170 ml/hour and 140 ml/hour, respectively. The working time of this stage is more than 7 hours starting from full LN2 and LHe tanks. Instrumental resolution of our cryo-stage cooled to 4.2 K was confirmed to be 0.20 nm by an optical diffraction pattern from the image of a chlorinated copper-phthalocyanine crystal. The image and the optical diffraction pattern are shown in Fig. 2 a, b, respectively.

Imaging of polymer single crystals in low-voltage, high-resolution scanning electron microscopy

1990 ◽  
Vol 48 (4) ◽  
pp. 1106-1107
Author(s):  
W.W. Adams ◽  
G. Price ◽  
A. Krause
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Single Crystals ◽  
Low Voltage ◽  
Polymer Surface ◽  
Beam Current ◽  
Image Features ◽  
First Results ◽  
Scanning Electron

It has been shown that there are numerous advantages in imaging both coated and uncoated polymers in scanning electron microscopy (SEM) at low voltages (LV) from 0.5 to 2.0 keV compared to imaging at conventional voltages of 10 to 20 keV. The disadvantages of LVSEM of degraded resolution and decreased beam current have been overcome with the new generation of field emission gun SEMs. In imaging metal coated polymers in LVSEM beam damage is reduced, contrast is improved, and charging from irregularly shaped features (which may be unevenly coated) is reduced or eliminated. Imaging uncoated polymers in LVSEM allows direct observation of the surface with little or no charging and with no alterations of surface features from the metal coating process required for higher voltage imaging. This is particularly important for high resolution (HR) studies of polymers where it is desired to image features 1 to 10 nm in size. Metal sputter coating techniques produce a 10 - 20 nm film that has its own texture which can obscure topographical features of the original polymer surface. In examining thin, uncoated insulating samples on a conducting substrate at low voltages the effect of sample-beam interactions on image formation and resolution will differ significantly from the effect at higher accelerating voltages. We discuss here sample-beam interactions in single crystals on conducting substrates at low voltages and also present the first results on HRSEM of single crystal morphologies which show some of these effects.

The Preparation of C60/C70 and the Characterization by Different Techniques

1992 ◽  
Vol 270 ◽  
Author(s):  
P. Häussler ◽  
H. Bestgen ◽  
H.-U. ter Meer
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Single Crystals ◽  
Absorption Spectra ◽  
Preparation Conditions ◽  
Transmission Electron ◽  
Method Evidence ◽  
Fcc Structure

ABSTRACTWe report on the preparation of C60/C70 by the arc method. Evidence of monofunctional C60O, and C60-CH2 has been observed by mass spectrometry. Dependent on their preparation conditions, high resolution transmission electron microscopy of crystals consisting of mixtures of C60/C70 show different structure. Needle-like crystals grown from toluene show superlattice formation while single crystals obtained by sublimation have fcc structure. UV-VIS-absorption spectra in toluene are compared to those of films of various thickness and crystallinity.

scholarly journals Simplified Approach for Preparing Graphene Oxide TEM Grids for Stained and Vitrified Biomolecules

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 643
Author(s):  
Anil Kumar ◽  
Nayanika Sengupta ◽  
Somnath Dutta
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Discharge System ◽  
Simplified Approach ◽  
Heavy Equipment ◽  
Peristaltic Pumps ◽  
High Resolution Structure

In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir–Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-H2O grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-H2O fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.

scholarly journals Electron Microscopy of Biological Macromolecules: Bridging the Gap between What Physics Allows and What We Currently Can Get

2004 ◽  
Vol 10 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Dieter Typke ◽  
Kenneth H. Downing ◽  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Low Dose ◽  
Carbon Film ◽  
Routine Data ◽  
Radiation Sensitivity ◽  
Similar Degree ◽  
Biological Macromolecules ◽  
Relative Ease ◽  
Thin Carbon

The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Because specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at ∼0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25%–35% of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.

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