Preparing Better Samples for Cryo–Electron Microscopy: Biochemical Challenges Do not End with Isolation and Purification

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Robert M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Effective Control ◽  
Annual Review ◽  
Publication Date ◽  
Biological Macromolecules ◽  
Isolation And Purification ◽  
Cryo Electron Microscopy ◽  
Extreme Risk ◽  
Resolution Electron

The preparation of extremely thin samples, which are required for high-resolution electron microscopy, poses extreme risk of damaging biological macromolecules due to interactions with the air-water interface. Although the rapid increase in the number of published structures initially gave little indication that this was a problem, the search for methods that substantially mitigate this hazard is now intensifying. The two main approaches under investigation are ( a) immobilizating particles onto structure-friendly support films and ( b) reducing the length of time during which such interactions may occur. While there is little possibility of outrunning diffusion to the interface, intentional passivation of the interface may slow the process of adsorption and denaturation. In addition, growing attention is being given to gaining more effective control of the thickness of the sample prior to vitrification. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals The endurance of flash-frozen biologics can be improved allowing high-resolution electron microscopy tomography on individual proteins

2021 ◽  
Author(s):  
Andrea Fera
Keyword(s):  
Electron Microscopy ◽  
Protein Interactions ◽  
Electron Beams ◽  
High Resolution Electron Microscopy ◽  
High Energy ◽  
Experimental Session ◽  
Protein Protein Interactions ◽  
Viral Origin ◽  
Cryo Electron Microscopy ◽  
Resolution Electron

Abstract Here surprising results are shown demonstrating a workflow possibly able to exploit the discreet nature of interactions between high-energy electron beams and matter. Isolated protein constructs have been imaged after an original temperature-curing in vacuum, introduced recently for flash-frozen rigid biopolymers, and here applied to flash-frozen oligomers of viral origin. These results, if confirmed, may extend to plastics and bio-oligomers the access to atomic coordinates in one experimental session, when imaged by electron microscopy. Which is the case when imaging semiconductors or other solid materials, provided that the samples are not damaged by the interaction with accelerated electron beams in vacuum. Therefore, potentially, this workflow introduces the possibility of achieving atomic resolution in only one experiment with data only about one individual protein, maybe out of thermodynamic equilibrium. Such data are vital to understand protein-protein interactions. Finally, this workflow offers the possibility, new to cryo-electron microscopy samples, to store a sample indefinitely under liquid nitrogen for imaging the same molecules in more than one experimental session.

Thin Carbon Film Areas Produced under Controlled Conditions

1973 ◽  
Vol 31 ◽  
pp. 78-79
Author(s):  
Nabil Rizk ◽  
Irwin Bendet
Keyword(s):  
Electron Microscopy ◽  
Carbon Film ◽  
High Resolution Electron Microscopy ◽  
Carbon Films ◽  
The Other ◽  
Biological Macromolecules ◽  
Resolution Electron ◽  
Thin Carbon ◽  
Carbon Substrates ◽  
Thin Carbon Film

The need for increasingly thin films to attain the ultimate in high resolution electron microscopy has seen the development of carbon substrates for the support of biological macromolecules. To date, the procedures employed for producing such substrates have involved either the floating off of carbon films from mica onto water and picking them up on EM grids or the deposition of carbon upon plastic covered grids, the plastic of which is subsequently dissolved away. We have now developed an efficient technique, unencumbered by the difficulties associated with the other procedures, for producing well delineated areas of thin carbon film of measurable thickness. In this procedure we utilize silk threads, about 15 microns in diameter, stretched across an adjustable frame having a 1” square opening (Fig. 1).

Recent Applications of High Resolution Electron Microscopy to Crystalline Arrays of Virus Particles

1978 ◽  
Vol 36 (3) ◽  
pp. 470-482 ◽  
Author(s):  
R.W. Horne
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Analytical Techniques ◽  
Staining Technique ◽  
High Resolution Electron Microscopy ◽  
Optical Diffraction ◽  
Full Potential ◽  
Virus Particles ◽  
Resolution Electron ◽  
Wide Range

The technique of surrounding virus particles with a neutralised electron dense stain was described at the Fourth International Congress on Electron Microscopy, Berlin 1958 (see Home & Brenner, 1960, p. 625). For many years the negative staining technique in one form or another, has been applied to a wide range of biological materials. However, the full potential of the method has only recently been explored following the development and applications of optical diffraction and computer image analytical techniques to electron micrographs (cf. De Hosier & Klug, 1968; Markham 1968; Crowther et al., 1970; Home & Markham, 1973; Klug & Berger, 1974; Crowther & Klug, 1975). These image processing procedures have allowed a more precise and quantitative approach to be made concerning the interpretation, measurement and reconstruction of repeating features in certain biological systems.

One Million Direct Magnification Microscopy

1971 ◽  
Vol 29 ◽  
pp. 166-167
Author(s):  
J. A. Hugo ◽  
V. A. Phillips
Keyword(s):  
Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Illumination Level ◽  
Level Of Detail ◽  
Photographic Emulsion ◽  
Low Contrast ◽  
Fluorescent Screen ◽  
Resolution Electron ◽  
Lattice Images ◽  
Electron Microscopes

A continuing problem in high resolution electron microscopy is that the level of detail visible to the microscopist while he is taking a picture is inferior to that obtainable by the microscope, readily readable on a photographic emulsion and visible in an enlargement made from the plate. Line resolutions, of 2Å or better are now achievable with top of the line 100kv microscopes. Taking the resolution of the human eye as 0.2mm, this indicates a need for a direct viewing magnification of at least one million. However, 0.2mm refers to optimum viewing conditions in daylight or the equivalent, and certainly does not apply to a (colored) image of low contrast and illumination level viewed on a fluorescent screen through a glass window by the dark-adapted eye. Experience indicates that an additional factor of 5 to 10 magnification is needed in order to view lattice images with line spacings of 2 to 4Å. Fortunately this is provided by the normal viewing telescope supplied with most electron microscopes.

Structural analysis of the surface-layer protein of spirillum serpens by high-resolution electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 738-739
Author(s):  
W. H. Wu ◽  
R. M. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Structural Analysis ◽  
High Resolution ◽  
Low Dose ◽  
High Resolution Electron Microscopy ◽  
Optical Diffraction ◽  
Surface Layer Protein ◽  
Morphological Unit ◽  
Resolution Electron

Spirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits. A morphological model of the structure of the protein has been proposed at a resolution of about 25 Å, in which the morphological unit might be described as having the appearance of a flared-out, hollow cylinder with six ÅspokesÅ at the flared end. In order to understand the detailed association of the macromolecules, it is necessary to do a high resolution structural analysis. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl2, a procedure derived from that of Buckmire and Murray. Low dose, low temperature electron microscopy has been applied to the large arrays.As a first step, the samples were negatively stained with neutralized phosphotungstic acid, and the specimens were imaged at 40,000 magnification by use of a high resolution cold stage on a JE0L 100B. Low dose images were recorded with exposures of 7-9 electrons/Å2. The micrographs obtained (Fig. 1) were examined by use of optical diffraction (Fig. 2) to tell what areas were especially well ordered.

Electron microscopy of rabbit FC crystals

1983 ◽  
Vol 41 ◽  
pp. 730-731
Author(s):  
Robert A. Grant ◽  
Laura L. Degn ◽  
Wah Chiu ◽  
John Robinson
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Resolution Electron Microscopy ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
Resolution Electron ◽  
Replacement Technique ◽  
Hydrated Crystal ◽  
Molecular Structure Analysis

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

Quality films from carbon fiber evaporation for thorough coverage of TEM grids

1988 ◽  
Vol 46 ◽  
pp. 418-419
Author(s):  
N. Tempel ◽  
M. C. Ledbetter
Keyword(s):  
Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Carbon Films ◽  
Low Noise ◽  
Vacuum Evaporation ◽  
High Yield ◽  
Good Adhesion ◽  
Noise Structure ◽  
Resolution Electron ◽  
Carbon Foils

Carbon films have been a support of choice for high resolution electron microscopy since the introduction of vacuum evaporation of carbon. The desirable qualities of carbon films and methods of producing them has been extensively reviewed. It is difficult to get a high yield of grids by many of these methods, especially if virtually all of the windows must be covered with a tightly bonded, quality film of predictable thickness. We report here a method for producing carbon foils designed to maximize these attributes: 1) coverage of virtually all grid windows, 2) freedom from holes, wrinkles or folds, 3) good adhesion between film and grid, 4) uniformity of film and low noise structure, 5) predictability of film thickness, and 6) reproducibility.Our method utilizes vacuum evaporation of carbon from a fiber onto celloidin film and grid bars, adhesion of the film complex to the grid by carbon-carbon contact, and removal of the celloidin by acetone dissolution. Materials must be of high purity, and cleanliness must be rigorously maintained.

High resolution electron microscopy of lanthanum phosphate crystals

1978 ◽  
Vol 36 (1) ◽  
pp. 274-275
Author(s):  
J. C. Wheatley ◽  
J. M. Cowley
Keyword(s):  
Electron Microscopy ◽  
Catalytic Activity ◽  
High Resolution ◽  
Petroleum Industry ◽  
High Resolution Electron Microscopy ◽  
Lanthanum Phosphate ◽  
Good Catalyst ◽  
Resolution Electron ◽  
Good Catalytic Activity ◽  
Physical And Chemical

Rare-earth phosphates are of particular interest because of their catalytic properties associated with the hydrolysis of many aromatic chlorides in the petroleum industry. Lanthanum phosphates (LaPO4) which have been doped with small amounts of copper have shown increased catalytic activity (1). However the physical and chemical characteristics of the samples leading to good catalytic activity are not known.Many catalysts are amorphous and thus do not easily lend themselves to methods of investigation which would include electron microscopy. However, the LaPO4, crystals are quite suitable samples for high resolution techniques.The samples used were obtained from William L. Kehl of Gulf Research and Development Company. The electron microscopy was carried out on a JEOL JEM-100B which had been modified for high resolution microscopy (2). Standard high resolution techniques were employed. Three different sample types were observed: 669A-1-5-7 (poor catalyst), H-L-2 (good catalyst) and 27-011 (good catalyst).

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