High-performance transmission electron microscopy of cryospecimens

Author(s):  
U. B. Hezel ◽  
E. Zellmann ◽  
D. Hoffmeister

Chemical fixation, resin embedding and subsequent staining with heavy metals can both produce artefacts and limit the resolution in the electron microscopic specimen /1-5/. The objective is thus to observe the specimens in the electron microscope in the frozen-hydrated state, the one most similar to the native state.All preparation steps such as cryofixation, cryosectioning and cryotransfer to the cryo-transmission electron microscope (CryoTEM) should be performed below 145K /5/ in order to observe the specimen in a matrix of vitrified ice avoiding any crystallization artefacts. In the CryoTEM itself the temperature of the frozen-hydrated specimen should be kept much lower to avoid devitrification caused by the electron beam /1/. - To meet all these requirements a special cryotransfer system and cryostage for the Zeiss transmission electron microscopes have been developed /7/.

1998 ◽  
Vol 4 (S2) ◽  
pp. 740-741
Author(s):  
J. Liu ◽  
J.R. Ebner

Catalyst characterization plays a vital role in new catalyst development and in troubleshooting of commercially catalyzed processes. The ultimate goal of catalyst characterization is to understand the structure-property relationships associated with the active components and supports. Among many characterization techniques, only electron microscopy and associated analytical techniques can provide local information about the structure, chemistry, morphology, and electronic properties of industrial heterogeneous catalysts. Three types of electron microscopes are usually used for characterizing industrial supported catalysts: 1) scanning electron microscope (SEM), 2) scanning transmission electron microscope (STEM), and 3) transmission electron microscope (TEM). Each type of microscope has its unique capabilities. However, the integration of all electron microscopic techniques has proved invaluable for extracting useful information about the structure and the performance of industrial catalysts.Commercial catalysts usually have a high surface area with complex geometric structures to enable reacting gases or fluids to access as much of the active surface of the catalyst as possible.


Author(s):  
R. A. Waugh ◽  
J. R. Sommer

Cardiac sarcoplasmic reticulum (SR) is a complex system of intracellular tubules that, due to their small size and juxtaposition to such electron-dense structures as mitochondria and myofibrils, are often inconspicuous in conventionally prepared electron microscopic material. This study reports a method with which the SR is selectively “stained” which facilitates visualizationwith the transmission electron microscope.


Author(s):  
G.K.W. Balkau ◽  
E. Bez ◽  
J.L. Farrant

The earliest account of the contamination of electron microscope specimens by the deposition of carbonaceous material during electron irradiation was published in 1947 by Watson who was then working in Canada. It was soon established that this carbonaceous material is formed from organic vapours, and it is now recognized that the principal source is the oil-sealed rotary pumps which provide the backing vacuum. It has been shown that the organic vapours consist of low molecular weight fragments of oil molecules which have been degraded at hot spots produced by friction between the vanes and the surfaces on which they slide. As satisfactory oil-free pumps are unavailable, it is standard electron microscope practice to reduce the partial pressure of organic vapours in the microscope in the vicinity of the specimen by using liquid-nitrogen cooled anti-contamination devices. Traps of this type are sufficient to reduce the contamination rate to about 0.1 Å per min, which is tolerable for many investigations.


Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.


Author(s):  
M.A. O’Keefe ◽  
J. Taylor ◽  
D. Owen ◽  
B. Crowley ◽  
K.H. Westmacott ◽  
...  

Remote on-line electron microscopy is rapidly becoming more available as improvements continue to be developed in the software and hardware of interfaces and networks. Scanning electron microscopes have been driven remotely across both wide and local area networks. Initial implementations with transmission electron microscopes have targeted unique facilities like an advanced analytical electron microscope, a biological 3-D IVEM and a HVEM capable of in situ materials science applications. As implementations of on-line transmission electron microscopy become more widespread, it is essential that suitable standards be developed and followed. Two such standards have been proposed for a high-level protocol language for on-line access, and we have proposed a rational graphical user interface. The user interface we present here is based on experience gained with a full-function materials science application providing users of the National Center for Electron Microscopy with remote on-line access to a 1.5MeV Kratos EM-1500 in situ high-voltage transmission electron microscope via existing wide area networks. We have developed and implemented, and are continuing to refine, a set of tools, protocols, and interfaces to run the Kratos EM-1500 on-line for collaborative research. Computer tools for capturing and manipulating real-time video signals are integrated into a standardized user interface that may be used for remote access to any transmission electron microscope equipped with a suitable control computer.


1987 ◽  
Vol 35 (8) ◽  
pp. 909-916 ◽  
Author(s):  
G D Gagne ◽  
M F Miller

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.


1986 ◽  
Vol 44 (4) ◽  
pp. 334-340 ◽  
Author(s):  
Cáudio A. Ferraz de Carvalho ◽  
II-Sei Watanabe ◽  
Romeu Rodrigues de Souza

Topographical variations of the ependymal surface of the whole brain ventricular system of Cebus apella, a wild Brazilian species, were studied at the scanning electron microscope after C02 critical point drying and carbon-gold coating. Certain selected areas were also studied at the transmission electron microscope, following standard technical procedures. We observed distinct patterns in the distribution of cilia, microvilli, other cell projections, and the so called supraependymal structures. Based on the morphological findings, functional considerations are made.


1997 ◽  
Vol 3 (S2) ◽  
pp. 1243-1244 ◽  
Author(s):  
Raynald Gauvin ◽  
Steve Yue

The observation of microstructural features smaller than 300 nm is generally performed using Transmission Electron Microscopy (TEM) because conventional Scanning Electron Microscopes (SEM) do not have the resolution to image such small phases. Since the early 1990’s, a new generation of microscopes is now available on the market. These are the Field Emission Gun Scanning Electron Microscope with a virtual secondary electron detector. The field emission gun gives a higher brightness than those obtained using conventional electron filaments allowing enough electrons to be collected to operate the microscope with incident electron energy, E0, below 5 keV with probe diameter smaller than 5 nm. At 1 keV, the electron range is 60 nm in aluminum and 10 nm in iron (computed using the CASINO program). Since the electron beam diameter is smaller than 5 nm at 1 keV, the resolution of these microscopes becomes closer to that of TEM.


2011 ◽  
Vol 19 (3) ◽  
pp. 10-14 ◽  
Author(s):  
Joerg R. Jinschek ◽  
Emrah Yucelen ◽  
Bert Freitag ◽  
Hector A. Calderon ◽  
Andy Steinbach

In his now-famous 1959 speech on nanotechnology, Richard Feynman proposed that it should be possible to see the individual atoms in a material, if only the electron microscope could be made 100 times better. With the development of aberration correctors on transmission electron microscopes (TEMs) over the last decade, this dream of microscopists to directly image structures atom-by-atom has come close to an everyday reality. Figure 1 shows such a high-resolution transmission electron microscope (HR-TEM) image of a single-wall carbon nanotube obtained with an aberration-corrected TEM. Now that atomic-resolution images have become possible with aberration-corrector technology in both TEM and STEM, we can ask ourselves if we truly have achieved the goal of seeing individual atoms. Most aberration-corrected images exhibiting atomic resolution are not distinguishing individual atoms, but columns of a small number of atoms, so despite this remarkable achievement, there is still “plenty of room at the bottom” in order to move toward seeing, counting, and quantifying individual atoms. In fact, there never has been a more exciting time for electron microscopists.


2020 ◽  
Vol 68 (6) ◽  
pp. 389-402
Author(s):  
Lars Möller ◽  
Gudrun Holland ◽  
Michael Laue

Diagnostic electron microscopy is a useful technique for the identification of viruses associated with human, animal, or plant diseases. The size of virus structures requires a high optical resolution (i.e., about 1 nm), which, for a long time, was only provided by transmission electron microscopes operated at 60 kV and above. During the last decade, low-voltage electron microscopy has been improved and potentially provides an alternative to the use of high-voltage electron microscopy for diagnostic electron microscopy of viruses. Therefore, we have compared the imaging capabilities of three low-voltage electron microscopes, a scanning electron microscope equipped with a scanning transmission detector and two low-voltage transmission electron microscopes, operated at 25 kV, with the imaging capabilities of a high-voltage transmission electron microscope using different viruses in samples prepared by negative staining and ultrathin sectioning. All of the microscopes provided sufficient optical resolution for a recognition of the viruses tested. In ultrathin sections, ultrastructural details of virus genesis could be revealed. Speed of imaging was fast enough to allow rapid screening of diagnostic samples at a reasonable throughput. In summary, the results suggest that low-voltage microscopes are a suitable alternative to high-voltage transmission electron microscopes for diagnostic electron microscopy of viruses.


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