A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freeze-substitution

1986 ◽  
Vol 4 (3) ◽  
pp. 177-240 ◽  
Author(s):  
Bert Ph. M. Menco
Keyword(s):  
Freeze Substitution ◽  
Freeze Fracturing ◽  
Freeze Etching

Cryo-SEM and TEM of High Pressure Frozen Cells - Some Technical Contributions

2001 ◽  
Vol 7 (S2) ◽  
pp. 728-729
Author(s):  
Paul Walther
Keyword(s):  
High Pressure ◽  
Physiological State ◽  
Electron Microscopic Observation ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Electron Microscopic ◽  
Biological Specimen ◽  
Freeze Fracturing

Imaging of fast frozen samples is the most direct approach for electron microscopy of biological specimen in a defined physiological state. It prevents chemical fixation and drying artifacts. High pressure freezing allows for ice-crystal-free cryo-fixation of tissue pieces up to a thickness of 200 urn and a diameter of 2 mm without prefixation. Such a frozen disc, however, is not directly amenable to electron microscopic observation: The structures of interest have to be made amenable to the electron beam, and the structures of interest must produce enough contrast to be recognized in the electron microscope. This can be achieved by freeze fracturing, cryo-sectioning or freeze substitution.The figures show high pressure frozen bakers yeast saccharomyces cerevisiae in the cryo-SEM (Figures 1 and 2) and after freeze substitution in the TEM (Figure 3). For high pressure freezing either a Bal-Tec HPM 010 (Princ. of Liechtenstein; Figures 1 and 2), or a Wohlwend HPF (Wohlwend GmbH, Sennwald, Switzerland; Figure 3) were used.

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.

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.

Mobility of Chloroplast Coupling Factor 1 (CF1) at the Thylakoid Surface as Revealed by Freeze-Etching after Antibody Labelling

1974 ◽  
Vol 29 (11-12) ◽  
pp. 694-699 ◽  
Author(s):  
Richard Berzborn ◽  
Friedrich Kopp ◽  
Kurt Mühlethaler
Keyword(s):  
Outer Surface ◽  
Coupling Factor ◽  
Freeze Fracturing ◽  
Deep Etching ◽  
Chloroplast Coupling Factor ◽  
Freeze Etching ◽  
Chloroplast Coupling Factor 1 ◽  
Number Of Particles ◽  
Antibody Labelling

Abstract Freeze-Etching Freeze-fracturing and 60 sec deep-etching of isolated chloroplast thylakoid systems exposed large areas of the outer surface (matrix side) of the thylakoids. If the thylakoid systems were first treated with antisera against chloroplast coupling factor 1 (CF1), the 14 nm particles at the outer surface appeared aggregated. Between clusters these particles were absent. Since there is no change in the number of particles/area after treatment with antibodies, it is concluded that the 14 nm particles are mobile within the surface of the thylakoid. The antisera contained only anti­bodies against CF1 ; therefore the 14 nm particles at the outer surface are identified to be CF1 . The implication of a mobile ATP-synthetase (CF1) for the mechanisms of photophosphorylation is discussed.

scholarly journals Role of Cell Shape in Determination of the Division Plane in Schizosaccharomyces pombe: Random Orientation of Septa in Spherical Cells

2000 ◽  
Vol 182 (6) ◽  
pp. 1693-1701 ◽  
Author(s):  
M. Sipiczki ◽  
M. Yamaguchi ◽  
A. Grallert ◽  
K. Takeo ◽  
E. Zilahi ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mother Cell ◽  
Freeze Substitution ◽  
Longitudinal Axis ◽  
Electron Microscopic ◽  
Division Plane ◽  
Daughter Cells ◽  
Freeze Etching ◽  
Spherical Cells

ABSTRACT The establishment of growth polarity in Schizosaccharomyces pombe cells is a combined function of the cytoplasmic cytoskeleton and the shape of the cell wall inherited from the mother cell. The septum that divides the cylindrical cell into two siblings is formed midway between the growing poles and perpendicularly to the axis that connects them. Since the daughter cells also extend at their ends and form their septa at right angles to the longitudinal axis, their septal (division) planes lie parallel to those of the mother cell. To gain a better understanding of how this regularity is ensured, we investigated septation in spherical cells that do not inherit morphologically predetermined cell ends to establish poles for growth. We studied four mutants (defining four novel genes), over 95% of whose cells displayed a completely spherical morphology and a deficiency in mating and showed a random distribution of cytoplasmic microtubules, Tea1p, and F-actin, indicating that the cytoplasmic cytoskeleton was poorly polarized or apolar. Septum positioning was examined by visualizing septa and division scars by calcofluor staining and by the analysis of electron microscopic images. Freeze-substitution, freeze-etching, and scanning electron microscopy were used. We found that the elongated bipolar shape is not essential for the determination of a division plane that can separate the postmitotic nuclei. However, it seems to be necessary for the maintenance of the parallel orientation of septa over the generations. In the spherical cells, the division scars and septa usually lie at angles to each other on the cell surface. We hypothesize that the shape of the cell indirectly affects the positioning of the septum by directing the extension of the spindle.

scholarly journals Inactivation of swmA Results in the Loss of an Outer Cell Layer in a Swimming Synechococcus Strain

2005 ◽  
Vol 187 (1) ◽  
pp. 224-230 ◽  
Author(s):  
J. McCarren ◽  
J. Heuser ◽  
R. Roth ◽  
N. Yamada ◽  
M. Martone ◽  
...  
Keyword(s):  
Cell Layer ◽  
Freeze Substitution ◽  
Outer Cell ◽  
Wild Type ◽  
Gene Encoding ◽  
Unique Type ◽  
Freeze Fracturing ◽  
Additional Layer ◽  
Synechococcus Strain ◽  
Outer Cell Layer

ABSTRACT The mechanism of nonflagellar swimming of marine unicellular cyanobacteria remains poorly understood. SwmA is an abundant cell surface-associated 130-kDa glycoprotein that is required for the generation of thrust in Synechococcus sp. strain WH8102. Ultrastructural comparisons of wild-type cells to a mutant strain in which the gene encoding SwmA has been insertionally inactivated reveal that the mutant lacks a layer external to the outer membrane. Cryofixation and freeze-substitution are required for the preservation of this external layer. Freeze fracturing and etching reveal that this additional layer is an S-layer. How the S-layer might function in motility remains elusive; however, this work describes an ultrastructural component required for this unique type of swimming. In addition, the work presented here describes the envelope structure of a model swimming cyanobacterium.

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