scholarly journals Freeze-fracturing in ultrahigh vacuum at -196 degrees C.

1978 ◽  
Vol 76 (3) ◽  
pp. 712-728 ◽  
Author(s):  
H Gross ◽  
E Bas ◽  
H Moor
Keyword(s):  
Freeze Fracture ◽  
Ultrahigh Vacuum ◽  
Conventional Technique ◽  
Optical Diffraction ◽  
Yeast Cells ◽  
Freeze Fracturing ◽  
Structural Distortions ◽  
Specimen Temperature ◽  
Freeze Etching ◽  
Diffraction Patterns

Conventional freeze-etching is carried out in a vacuum of approximately 10(-6) torr and at a specimen temperature of -100 degrees C. The relatively poor topographic resolution of most freeze-etch replicas, and the lack of complementarity of morphological details in double replicas have been thought to be caused by structural distortions during fracturing, and radiation damage during replication. Both phenomena can be reduced by lowering the specimen temperature. To prevent condensation of residual gases (especially H2O) on the fracture faces at lower specimen temperature, an improved vacuum is required. Therefore, an ultrahigh vacuum freeze-fracture apparatus has been developed which allows fracturing and Pt/C-shadowing of specimens at -196 degrees C while maintaining a vacuum of 10(-9) torr. It consists of a modified Balzers BA 350 ultrahigh vacuum (UHV) unit, equipped with an airlock which enables the input of nonhoar-frosted specimens directly into the evacuated bell jar. A comparison of the paracrystalline plasmalemma structure in yeast cells portrayed by the conventional technique and by UHV-freeze-fracturing at -196 degrees C shows the improved topographic resolution which has been achieved with the new technique. The improvement is explained by less structural distortions during fracturing at lower temperatures. The particles of the paracrystalline regions on the P face are more regularly arranged and exhibit a craterlike substructure which corresponds with a ringlike depression in the E face. The optical diffraction patterns of these paracrystalline regions demonstrate the improvement of the structural record by showing well-defined third- and fourth-order spots.

scholarly journals Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

1978 ◽  
Vol 79 (1) ◽  
pp. 276-280 ◽  
Author(s):  
UB Sleytr ◽  
P Messner
Keyword(s):  
Liquid Nitrogen ◽  
Freeze Fracture ◽  
Ultrahigh Vacuum ◽  
Drastic Reduction ◽  
Preparation Conditions ◽  
Vacuum Technology ◽  
Significant Difference ◽  
Specimen Area ◽  
Freeze Etching ◽  
Regular Arrays

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.

Low temperature SEM comparisons of adjacent freeze-fractured and freeze-etched areas on frozen, hydrated samples

1995 ◽  
Vol 53 ◽  
pp. 1066-1067
Author(s):  
William P. Wergin ◽  
Eric F. Erbe ◽  
Robert W. Yaklich
Keyword(s):  
Low Temperature ◽  
Biological Samples ◽  
Freeze Fracture ◽  
Structural Components ◽  
Freeze Fracturing ◽  
Water Ice ◽  
Virus Particles ◽  
Structural Details ◽  
Freeze Etching ◽  
Sem Imaging

Most biological samples contain 70-95% water, consequently cryofixation and freeze-fracturing result in relatively smooth surfaces that exhibit few structural details. Freeze-etching, a technique that solved this problem, was initially developed for TEM observations of virus particles by Steere nearly 40 years ago. The technique, which sublimes water-ice from the surface of a fractured sample, produces surface topography that corresponds to the structural components on the freeze-etched face. This technique was further enhanced by recovering the complementary halves of a fractured sample, etching one of the surfaces and then comparing the complementary replicas from the freeze-fractured and freeze-etched faces. Recently, similar techniques were used on frozen, hydrated samples to examine complementary halves of freeze-fractured, freeze-etched specimens by low temperature SEM. Imaging complementary images of frozen, hydrated specimens in the SEM was faster than imaging complementary replicas in the TEM, however the procedure required specialized holders and was technically demanding.To simplify comparisons of freeze-fracture, freeze-etch images, samples were frozen, fractured and etched in the prechamber of an Oxford CT 1500 HF Cryotrans system that was attached to a Hitachi S-4100 FESEM.

Light Optical Diffraction Studies of Macromolecular Ultrastructure

1970 ◽  
Vol 28 ◽  
pp. 258-259
Author(s):  
Glen B. Haydon
Keyword(s):  
High Resolution ◽  
Diffraction Analysis ◽  
Diffraction Pattern ◽  
Electron Micrographs ◽  
Optical Diffraction ◽  
Electron Micrograph ◽  
Its Analysis ◽  
Resolution Electron ◽  
Diffraction Patterns

Analysis of light optical diffraction patterns produced by electron micrographs can easily lead to much nonsense. Such diffraction patterns are referred to as optical transforms and are compared with transforms produced by a variety of mathematical manipulations. In the use of light optical diffraction patterns to study periodicities in macromolecular ultrastructures, a number of potential pitfalls have been rediscovered. The limitations apply to the formation of the electron micrograph as well as its analysis.(1) The high resolution electron micrograph is itself a complex diffraction pattern resulting from the specimen, its stain, and its supporting substrate. Cowley and Moodie (Proc. Phys. Soc. B, LXX 497, 1957) demonstrated changing image patterns with changes in focus. Similar defocus images have been subjected to further light optical diffraction analysis.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

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.

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.

Crystalline order to a resolution of 4.7 A in the sheath of Methanospirilium hungatii: A cross-beta structure

1984 ◽  
Vol 42 ◽  
pp. 214-215
Author(s):  
Murray Stewart ◽  
T.J. Beveridge ◽  
D. Sprott
Keyword(s):  
Cell Wall ◽  
Single Layer ◽  
Uranyl Acetate ◽  
Optical Diffraction ◽  
Tube Axis ◽  
Basic Subunit ◽  
Beta Structure ◽  
Diffraction Patterns ◽  
Protein Treatment ◽  
General Appearance

The archaebacterium Methanospirillum hungatii has a sheath as part of its cell wall which is composed mainly of protein. Treatment with dithiothreitol or NaOH released the intact sheaths and electron micrographs of this material negatively stained with uranyl acetate showed flattened hollow tubes, about 0.5 μm diameter and several microns long, in which the patterns from the top and bottom were superimposed. Single layers, derived from broken tubes, were also seen and were more simply analysed. Figure 1 shows the general appearance of a single layer. There was a faint axial periodicity at 28.5 A, which was stronger at irregular multiples of 28.5 A (3 and 4 times were most common), and fine striations were also seen at about 3° to the tube axis. Low angle electron diffraction patterns (not shown) and optical diffraction patterns (Fig. 2) from these layers showed a complex meridian (as a result of the irregular nature of the repeat along the tube axis) which showed a clear maximum at 28.5 A, consistent with the basic subunit spacing.

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

Three-Dimensional Immunoelectron Microscopy of Yeast Cells by a New High-Resolution Replica Method

1985 ◽  
Vol 43 ◽  
pp. 562-563
Author(s):  
Hirano T. ◽  
M. Yamaguchi ◽  
M. Hayashi ◽  
Y. Sekiguchi ◽  
A. Tanaka
Keyword(s):  
High Resolution ◽  
Plasma Polymerization ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Replica Method ◽  
Yeast Cells ◽  
Dynamic Aspect ◽  
Replica Technique ◽  
Gaseous Hydrocarbon ◽  
Transmission Electron

A plasma polymerization film replica method is a new high resolution replica technique devised by Tanaka et al. in 1978. It has been developed for investigation of the three dimensional ultrastructure in biological or nonbiological specimens with the transmission electron microscope. This method is based on direct observation of the single-stage replica film, which was obtained by directly coating on the specimen surface. A plasma polymerization film was deposited by gaseous hydrocarbon monomer in a glow discharge.The present study further developed the freeze fracture method by means of a plasma polymerization film produces a three dimensional replica of chemically untreated cells and provides a clear evidence of fine structure of the yeast plasma membrane, especially the dynamic aspect of the structure of invagination (Figure 1).

Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose

1989 ◽  
Vol 47 ◽  
pp. 1024-1025
Author(s):  
George C. Ruben ◽  
William Krakow
Keyword(s):  
Single Molecule ◽  
Helical Structure ◽  
Optical Diffraction ◽  
Maximum Diameter ◽  
Primary Cell Wall ◽  
Cellulose Synthesis ◽  
Left Hand ◽  
Left Handed ◽  
Freeze Dried ◽  
Diffraction Patterns

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.

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