An improved specimen table for the Balzers freeze etching system BAF 400

1982 ◽  
Vol 127 (2) ◽  
pp. 227-231 ◽  
Author(s):  
D. M. Wisse ◽  
F. Spies
Keyword(s):  
Freeze Etching ◽  
Etching System

Comparison of Acrylamide and Agar Gels by Freeze-Etching

1977 ◽  
Vol 35 ◽  
pp. 606-607
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Electron Microscope ◽  
Sodium Hydroxide ◽  
Sodium Hypochlorite ◽  
Chromic Acid ◽  
Distilled Water ◽  
Thin Sheets ◽  
Acid Cleaning ◽  
Agar Gels ◽  
Freeze Etching ◽  
Water Cut

Thin sheets of acrylamide and agar gels of different concentrations were prepared and washed in distilled water, cut into pieces of appropriate size to fit into complementary freeze-etch specimen holders (1) and rapidly frozen. Freeze-etching was accomplished in a modified Denton DFE-2 freeze-etch unit on a DV-503 vacuum evaporator.* All samples were etched for 10 min. at -98°C then re-cooled to -150°C for deposition of Pt-C shadow- and C replica-films. Acrylamide gels were dissolved in Chlorox (5.251 sodium hypochlorite) containing 101 sodium hydroxide, whereas agar gels dissolved rapidly in the commonly used chromic acid cleaning solutions. Replicas were picked up on grids with thin Foimvar support films and stereo electron micrographs were obtained with a JEM-100 B electron microscope equipped with a 60° goniometer stage.Characteristic differences between gels of different concentrations (Figs. 1 and 2) were sufficiently pronounced to convince us that the structures observed are real and not the result of freezing artifacts.

New Dimensions in Freeze-Etching

1969 ◽  
Vol 27 ◽  
pp. 202-203 ◽  
Author(s):  
Russell L. Steere ◽  
Michael Moseley
Keyword(s):  
Fracture Surfaces ◽  
Aluminum Specimen ◽  
Specimen Holder ◽  
Freeze Etching ◽  
The Right

A redesigned specimen holder and cap have made possible the freeze-etching of both fracture surfaces of a frozen fractured specimen. In principal, the procedure involves freezing a specimen between two specimen holders (as shown in A, Fig. 1, and the left side of Fig. 2). The aluminum specimen holders and brass cap are constructed so that the upper specimen holder can be forced loose, turned over, and pressed down firmly against the specimen stage to a position represented by B, Fig. 1, and the right side of Fig. 2.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

Evaluation of Freezing Methods by Low Temperature X-Ray Diffraction

1977 ◽  
Vol 35 ◽  
pp. 330-333
Author(s):  
T. Gulik-Krzywicki ◽  
M.J. Costello
Keyword(s):  
Biological Materials ◽  
Molecular Organization ◽  
X Ray Diffraction ◽  
Glass Capillary ◽  
X Ray ◽  
Rate Dependent ◽  
Before And After ◽  
Freeze Etching ◽  
Diffraction Patterns ◽  
Set Up

Freeze-etching electron microscopy is currently one of the best methods for studying molecular organization of biological materials. Its application, however, is still limited by our imprecise knowledge about the perturbations of the original organization which may occur during quenching and fracturing of the samples and during the replication of fractured surfaces. Although it is well known that the preservation of the molecular organization of biological materials is critically dependent on the rate of freezing of the samples, little information is presently available concerning the nature and the extent of freezing-rate dependent perturbations of the original organizations. In order to obtain this information, we have developed a method based on the comparison of x-ray diffraction patterns of samples before and after freezing, prior to fracturing and replication.Our experimental set-up is shown in Fig. 1. The sample to be quenched is placed on its holder which is then mounted on a small metal holder (O) fixed on a glass capillary (p), whose position is controlled by a micromanipulator.

Structures Formed by Cryoprotective Agents Used in Freezc-Etching

1971 ◽  
Vol 29 ◽  
pp. 448-449
Author(s):  
G. G. Cocks ◽  
C. E. Cluthe
Keyword(s):  
Aqueous Solution ◽  
Polyethylene Oxide ◽  
Liquid Nitrogen Temperature ◽  
Ice Crystals ◽  
Etching Technique ◽  
Structural Constituent ◽  
Cryoprotective Agents ◽  
Quick Freezing ◽  
Freeze Etching ◽  
Fractured Surface

The freeze etching technique is potentially useful for examining dilute solutions or suspensions of macromolecular materials. Quick freezing of aqueous solutions in Freon or propane at or near liquid nitrogen temperature produces relatively large ice crystals and these crystals may damage the structures to be examined. Cryoprotective agents may reduce damage to the specimem, hut their use often results in the formation of a different set of specimem artifacts.In a study of the structure of polyethylene oxide gels glycerol and sucrose were used as cryoprotective agents. The experiments reported here show some of the structures which can appear when these cryoprotective agents are used.Figure 1 shows a fractured surface of a frozen 25% aqueous solution of sucrose. The branches of dendritic ice crystals surrounded hy ice-sucrose eutectic can be seen. When this fractured surface is etched the ice in the dendrites sublimes giving the type of structure shown in Figure 2. The ice-sucrose eutectic etches much more slowly. It is the smooth continuous structural constituent surrounding the branches of the dendrites.

Freeze-Fracture Replication of Frozen Outer Segments of Rat Retinal Rods

1969 ◽  
Vol 27 ◽  
pp. 392-393
Author(s):  
Thomas S. Leeson ◽  
C. Roland Leeson
Keyword(s):  
Electron Microscopy ◽  
Cross Section ◽  
Outer Segment ◽  
Freeze Fracture ◽  
X Ray Diffraction ◽  
X Ray ◽  
Outer Segments ◽  
Retinal Rods ◽  
Temperature Electron ◽  
Freeze Etching

Numerous previous studies of outer segments of retinal receptors have demonstrated a complex internal structure of a series of transversely orientated membranous lamellae, discs, or saccules. In cones, these lamellae probably are invaginations of the covering plasma membrane. In rods, however, they appear to be isolated and separate discs although some authors report interconnections and some continuities with the surface near the base of the outer segment, i.e. toward the inner segment. In some species, variations have been reported, such as longitudinally orientated lamellae and lamellar whorls. In cross section, the discs or saccules show one or more incisures. The saccules probably contain photolabile pigment, with resulting potentials after dipole formation during bleaching of pigment. Continuity between the lamina of rod saccules and extracellular space may be necessary for the detection of dipoles, although such continuity usually is not found by electron microscopy. Particles on the membranes have been found by low angle X-ray diffraction, by low temperature electron microscopy and by freeze-etching techniques.

Characterization of photosystem II with the atomic-force microscope

1992 ◽  
Vol 50 (2) ◽  
pp. 1146-1147
Author(s):  
Kathleen M. Marr ◽  
Mary K. Lyon
Keyword(s):  
Photosystem Ii ◽  
Atomic Force Microscope ◽  
Three Dimensional ◽  
Biologically Active ◽  
Atomic Force ◽  
Freeze Etching ◽  
Image Averaging ◽  
Oxygen Evolving ◽  
Averaging Techniques

Photosystem II (PSII) is different from all other reaction centers in that it splits water to evolve oxygen and hydrogen ions. This unique ability to evolve oxygen is partly due to three oxygen evolving polypeptides (OEPs) associated with the PSII complex. Freeze etching on grana derived insideout membranes revealed that the OEPs contribute to the observed tetrameric nature of the PSIl particle; when the OEPs are removed, a distinct dimer emerges. Thus, the surface of the PSII complex changes dramatically upon removal of these polypeptides. The atomic force microscope (AFM) is ideal for examining surface topography. The instrument provides a topographical view of individual PSII complexes, giving relatively high resolution three-dimensional information without image averaging techniques. In addition, the use of a fluid cell allows a biologically active sample to be maintained under fully hydrated and physiologically buffered conditions. The OEPs associated with PSII may be sequentially removed, thereby changing the surface of the complex by one polypeptide at a time.

Freeze-fracture cytochemistry: A goal set by Russell Steere in 1957

1993 ◽  
Vol 51 ◽  
pp. 60-61
Author(s):  
Nicholas J Severs
Keyword(s):  
Chemical Nature ◽  
Biological Systems ◽  
Freeze Fracture ◽  
Structural Components ◽  
Major Goal ◽  
Membrane Biology ◽  
Routine Application ◽  
The Future ◽  
Freeze Etching

In his pioneering demonstration of the potential of freeze-etching in biological systems, Russell Steere assessed the future promise and limitations of the technique with remarkable foresight. Item 2 in his list of inherent difficulties as they then stood stated “The chemical nature of the objects seen in the replica cannot be determined”. This defined a major goal for practitioners of freeze-fracture which, for more than a decade, seemed unattainable. It was not until the introduction of the label-fracture-etch technique in the early 1970s that the mould was broken, and not until the following decade that the full scope of modern freeze-fracture cytochemistry took shape. The culmination of these developments in the 1990s now equips the researcher with a set of effective techniques for routine application in cell and membrane biology.Freeze-fracture cytochemical techniques are all designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied.

Rapid Freezing of Freeze-Etch Specimens

1980 ◽  
Vol 38 ◽  
pp. 752-755
Author(s):  
A. Elgsaeter ◽  
T. Espevik ◽  
G. Kopstad
Keyword(s):  
Low Temperature ◽  
Metal Surface ◽  
Relative Velocity ◽  
Thermal Contact ◽  
High Rate ◽  
Rapid Freezing ◽  
Temperature Decrease ◽  
Freeze Etching

The importance of a high rate of temperature decrease (“rapid freezing”) when freezing specimens for freeze-etching has long been recognized1. The two basic methods for achieving rapid freezing are: 1) dropping the specimen onto a metal surface at low temperature, 2) bringing the specimen instantaneously into thermal contact with a liquid at low temperature and subsequently maintaining a high relative velocity between the liquid and the specimen. Over the last couple of years the first method has received strong renewed interest, particularily as the result of a series of important studies by Heuser and coworkers 2,3. In this paper we will compare these two freezing methods theoretically and experimentally.

Rate of Sublimation of Ice by Radiative Heating in Freeze-Etching

1980 ◽  
Vol 38 ◽  
pp. 618-619
Author(s):  
Yeshayahu Talmon
Keyword(s):  
Heat Flux ◽  
Freeze Fracture ◽  
Constant Heat Flux ◽  
Radiative Heating ◽  
Constant Heat ◽  
Entire Sample ◽  
Simplified Method ◽  
Freeze Etching ◽  
Sublimation Rates ◽  
Fractured Surface

To bring out details in the fractured surface of a frozen sample in the freeze fracture/freeze-etch technique,the sample or part of it is warmed to enhance water sublimation.One way to do this is to raise the temperature of the entire sample to about -100°C to -90°C. In this case sublimation rates can be calculated by using plots such as Fig.1 (Talmon and Thomas),or by simplified formulae such as that given by Menold and Liittge. To achieve higher rates of sublimation without heating the entire sample a radiative heater can be used (Echlin et al.). In the present paper a simplified method for the calculation of the rates of sublimation under a constant heat flux F [W/m2] at the surface of the sample from a heater placed directly above the sample is described.

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