scholarly journals Freezing Techniques: History, Comparisons, and Applications

2008 ◽  
Vol 16 (5) ◽  
pp. 12-17 ◽  
Author(s):  
Bill Graham ◽  
Jotham R. Austin ◽  
Andres Kaech ◽  
John E. Heuser
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Basic Problem ◽  
High Vacuum ◽  
Harsh Environment ◽  
Specimen Preparation ◽  
Biological Processes ◽  
Thin Sectioning ◽  
The Cost ◽  
Preparation Techniques

Specimen preparation techniques have evolved hand in hand with microscopy since the first microscopes. Since the introduction of the first Electron Microscope (EM) in the 1930’s, the basic problem with biological electron microscopy has been to preserve the structure of soft condensed, hydrated matter (e.g. tissues, cells, proteins, etc.) so that they can be viewed in the harsh environment of the electron microscope's high vacuum and ionizing radiation. For this, cells must be “fixed” with chemical cross-linkers, commonly glutaraldehyde, formaldehyde or some combination of both, stained with heavy metals (osmium tetroxide that provides contrast of biological components), dehydrated with an organic solvent, and infiltrated with a resin for eventual thin-sectioning. Only then can it be viewed with the EM. Such treatment with chemical fixatives and stains remains the standard approaches to arrest biological processes in cells or tissues, but at the cost of introducing clearly recognizable artifacts.

scholarly journals A Versatile and Affordable Plunge Freezing Instrument for Preparing Frozen Hydrated Specimens for Cryo Transmission Electron Microscopy (CryoEM)

2009 ◽  
Vol 17 (2) ◽  
pp. 14-17 ◽  
Author(s):  
Linda Melanson
Keyword(s):  
Electron Microscopy ◽  
High Vacuum ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Preservation Method ◽  
Transmission Electron ◽  
Structural Details ◽  
Soft Polymer ◽  
2D Crystals ◽  
Preparation Techniques

CryoEM is a powerful tool in the arsenal of structural biologists and soft polymer chemists. Hydrated specimens require a preservation method that will counteract the effects of the electron beam and the high vacuum environment of the electron microscope. Classical specimen preparation techniques using chemical fixatives are not able to capture the native structure of the once hydrated specimen perfectly. In contrast to classical methods for preserving specimens for electron microscopy, rapid freezing of radiation-sensitive specimens such as dispersed biological macromolecular assemblies, 2D crystals, and colloids allows the structural details of the specimen to be captured in their essentially native state to near atomic resolution.

The ionic strength dependence of chromatin folding

1978 ◽  
Vol 36 (2) ◽  
pp. 220-221
Author(s):  
F. Thoma ◽  
TH. Koller
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Ionic Strength ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Carbon Supports ◽  
Ionic Strength Dependence ◽  
Chromatin Folding ◽  
Strength Dependence ◽  
Preparation Techniques

Under a variety of electron microscope specimen preparation techniques different forms of chromatin appearance can be distinguished: beads-on-a-string, a 100 Å nucleofilament, a 250 Å fiber and a compact 300 to 500 Å fiber.Using a standardized specimen preparation technique we wanted to find out whether there is any relation between these different forms of chromatin or not. We show that with increasing ionic strength a chromatin fiber consisting of a row of nucleo- somes progressively folds up into a solenoid-like structure with a diameter of about 300 Å.For the preparation of chromatin for electron microscopy the avoidance of stretching artifacts during adsorption to the carbon supports is of utmost importance. The samples are fixed with 0.1% glutaraldehyde at 4°C for at least 12 hrs. The material was usually examined between 24 and 48 hrs after the onset of fixation.

The Current Situation in Chemical Stabilization of Biological Materials

1972 ◽  
Vol 30 ◽  
pp. 132-133
Author(s):  
John H. Luft
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Molecular Level ◽  
Cross Linking ◽  
Chemical Stabilization ◽  
Specimen Preparation ◽  
Sufficient Condition ◽  
Radio Telescopes

With information processing devices such as radio telescopes, microscopes or hi-fi systems, the quality of the output often is limited by distortion or noise introduced at the input stage of the device. This analogy can be extended usefully to specimen preparation for the electron microscope; fixation, which initiates the processing sequence, is the single most important step and, unfortunately, is the least well understood. Although there is an abundance of fixation mixtures recommended in the light microscopy literature, osmium tetroxide and glutaraldehyde are favored for electron microscopy. These fixatives react vigorously with proteins at the molecular level. There is clear evidence for the cross-linking of proteins both by osmium tetroxide and glutaraldehyde and cross-linking may be a necessary if not sufficient condition to define fixatives as a class.

Electron Microscopy of Bacteriophage T7 Internal Head Proteins

1975 ◽  
Vol 33 ◽  
pp. 638-639
Author(s):  
P. Serwer
Keyword(s):  
Electron Microscopy ◽  
Bacteriophage T7 ◽  
Specimen Preparation ◽  
Dna Molecule ◽  
The Core ◽  
Internal Core ◽  
Phage T7 ◽  
T7 Phage ◽  
Preparation Techniques ◽  
Internal Head

The genome of bacteriophage T7 is a duplex DNA molecule packaged in a space whose volume has been measured to be 2.2 x the volume of the B form of T7 DNA. To help determine the mechanism for packaging this DNA, the configuration of proteins inside the phage head has been investigated by electron microscopy. A core which is roughly cylindrical in outline has been observed inside the head of phage T7 using three different specimen preparation techniques.When T7 phage are treated with glutaraldehyde, DNA is ejected from the head often revealing an internal core (dark arrows in Fig. 1). When both the core and tail are present in a particle, the core appears to be coaxial with the tail. Core-tail complexes sometimes dislodge from their normal location and appear attached to the outside of a phage head (light arrow in Fig. 1).

scholarly journals Vapor Coating: A Simple, Economical Procedure for Preparing Difficult Specimens for Scanning Electron Microscopy

2007 ◽  
Vol 15 (3) ◽  
pp. 44-45 ◽  
Author(s):  
E. Ann Ellis ◽  
Michael W. Pendleton
Keyword(s):  
Osmium Tetroxide ◽  
Petri Dish ◽  
Specimen Preparation ◽  
Fume Hood ◽  
Imaging Center ◽  
User Facility ◽  
Small Container ◽  
Set Up ◽  
Preparation Techniques ◽  
Scanning Electron

The Microscopy and Imaging Center at Texas A&M University is a multi-user facility involved with preparation and analysis of many different biological and materials sciences projects. Vapor stabilization and coating is an important part of our specimen preparation methodology for difficult biological and materials, especially polymer, samples. The procedure for all our vapor preparation techniques is done in a simple, economical apparatus set up in a properly functioning fume hood with a flow rate of at least 100 ft/min (Fig. 1). The apparatus is made from a glass petri dish or a glass petri dish for the bottom and an appropriate size beaker for the top. Specimens, mounted on stubs, are placed inside the chamber and the fixative (osmium tetroxide, ruthenium tetroxide or acrolein) is placed in a small container (plastic bottle cap) near the specimens.

Ultra-high-vacuum, high-resolution Transmission Electron Microscopy at 400 kV

1986 ◽  
Vol 44 ◽  
pp. 606-609
Author(s):  
F. A. Ponce ◽  
S. Suzuki ◽  
H. Kobayashi ◽  
Y. Ishibashi ◽  
Y. Ishida ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Vacuum ◽  
Milling Process ◽  
Electron Bombardment ◽  
Ultra High Vacuum ◽  
Specimen Preparation ◽  
Ion Milling ◽  
Transmission Electron ◽  
Preparation Stage ◽  
Resolution Imaging

Electron microscopy in an ultra high vacuum (UHV) environment is a very desirable capability for the study of surfaces and for near-atomic-resolution imaging. The existence of amorphous layers on the surface of the sample generally prevents the direct observation of the free surface structure and limits the degree of resolution in the transmission electron microscope (TEM). In conventional TEM, these amorphous layers are often of organic nature originating from the electron bombardment of hydrocarbons in the vicinity of the sample. They can in part also be contaminants which develop during the specimen preparation and transport stages. In the specimen preparation stage, contamination can occur due to backsputtering during the ion milling process. In addition, oxide layers develop from contact to air during transport to the TEM. In order to avoid these amorphous overlayers it is necessary: i) to improve the vacuum of the instrument, thus the need for ultra high vacuum; and ii) to be able to clean the sample and transfer it to the column of the instrument without breaking the vacuum around the sample.

Unconventional Specimen Preparation Techniques Using High Resolution Low Voltage Field Emission Scanning Electron Microscopy to Study Cell Motility, Host Cell Invasion, and Internal Cell Structures in Toxoplasma gondii

2002 ◽  
Vol 8 (2) ◽  
pp. 94-103 ◽  
Author(s):  
Heide Schatten ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Host Cell ◽  
Cell Invasion ◽  
Low Voltage ◽  
Cytoskeletal Proteins ◽  
Specimen Preparation ◽  
Host Cell Invasion ◽  
Apicomplexan Parasites ◽  
Preparation Techniques

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.

Development of high-vacuum equipment for EM specimen preparation

1992 ◽  
Vol 50 (2) ◽  
pp. 1082-1083
Author(s):  
Richard A. Denton
Keyword(s):  
Electron Microscopy ◽  
High Vacuum ◽  
The Other ◽  
Specimen Preparation ◽  
Vacuum Equipment ◽  
Optical Film ◽  
Diffusion Pump ◽  
Shadow Casting ◽  
Mechanical Pump ◽  
The U.S

High-vacuum techniques made electron microscopy possible. In the 1930s vacuum evaporators with glass or metal chambers, diffusion pumps and oil sealed mechanical pumps were used in Europe and the U.S. The earliest systems used mercury pumps with liquid air traps. Oil diffusion pumps were manufactured in the U.S. by D.P.I. from glass or metal. In 1940 the first RCA TEM went into production as the EMB. First shadow casting in the U.S. was by Williams and Wycoff in 1944 and in Europe by Műller in 1942. Due to war secrecy, neither knew about the other. In 1944 RCA built the first production evaporator for EM under the direction of Bob Picard. The system had an 18" dia. glass bell jar and a metal baseplate with an oil diffusion pump backed by a Cenco Hypervac 20 mechanical pump. In 1948 Optical Film Engineering designed a 12" dia. bell jar evaporator for EM. This SC-3 employed a Welch 5 cfm mechanical pump and a 3" diffusion pump. Carbon evaporation for substrates or replicas was invented by D.E. Bradley in England and published in 1954.

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