Freeze-etching: freezing velocity and crystal size at different locations in samples.
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Crystallography↗
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Improved cryofixation applicable to freeze etching.
Freeze etching of solute model systems (e.g., glycerol or ferritin solutions) demonstrates that cryofixation can introduce serious artifacts due to the segregation of the dissolved or dispersed material from the solvent. Since, in principle, this problem can be reduced by increasing the cooling rate, a new technique has been developed which combines spray freezing with freeze etching. This spray-freeze-etching is applied by first spraying the specimen into a liquid cryomedium. The frozen droplets are then "glued" together with butylbenzene to form a regular freeze-etch specimen, while the temperature of the sample is kept at -85 degrees C. The results obtained by spray-freeze-etching are far superior to those obtained by standard freezing. Our results, using 5% glycerol as a test specimen, are equivalent to those obtained by the high-pressure method (1). The reduction of segregation during freezing makes freeze etching a method applicable for the investigation of solute systems. Furthermore, the study of unicellular organisms or cellular fractions by freeze etching without the use of antifreeze is made possible.
Freeze Etching↗
Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure.
In order to test the ability of freeze substitution to accurately preserve the ultrastructure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical-point-drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical-point-dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right-handed) two start helix was not affected by the different methods of preparation. However, the left-handed "genetic" helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical-point-drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical-point-dried, filaments appeared to intersect at greater angles and seemed more "taut." These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing steps.
Actins↗
A new freeze-etch unit for freeze-etch rotary shadowing, low temperature freeze-fracturing and conventional freeze-etching.
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Brevibacterium↗
Fracture faces in intact cells and protoplasts of Bacillus stearothermophilus. A study by conventional freeze-etching and freeze-etching of corresponding fracture moieties.
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Bacillus↗
Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution.
The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4-6 nm in diameter. Fine filaments 8-10 nm in diameter and 30-50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3-5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.
Animals↗
A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation.
The ultrastructure of bull and boar spermatozoa was investigated following different cryopreparation methods and chemical fixation. Spray-freezing was used for cryofixation in both freeze-etching and freeze-drying studies. Freeze-etching of boar spermatozoa revealed that the arrangement of the postnuclear striations differed from that in the bull. Freeze-drying gave excellent results for structural preservation, which were equal to those of chemical fixation. Some structural details not visible in chemically fixed cells were detected in freeze-dried and vacuum-embedded bull and boar spermatozoa, e.g. the arrangement of the lamellar nuclear contents, known from freeze-fractures, and a fine lamellar structure of the acrosomal contents. Cryofixation by spray-freezing combined with freeze-drying makes any contact of the cells with fixatives, buffer solutions and dehydration media unnecessary, and potentially provides all the advantages of ultrathin sectioning required for histochemical studies.
Acrosome↗
[The freeze-etching and freeze-fracturing technics and their use for studying cellular membranes in the central nervous system].
In the present review, both subkinds of the method for membrane splitting, namely freeze-etching and freeze-fracturing techniques, are presented. After a brief historical survey, a mechanism of their action is described. Special attention is paid to the consecutive stages in applying these techniques--fixation, fracturing, etching, replication, cleaning and observation. A new idea for a better cleaning of the replicas and an explanation of the different occurrence of intramembranous particles on the membrane fracture faces are proposed. Finally, the application of the freeze-etching and freeze-fracturing techniques to visualize the intramembranous structure of membranes in the central nervous system is outlined.
Animals↗
Freeze-etching and freeze-fracture structural features of cell envelopes in mycobacteria and leprosy derived corynebacteria.
The structural properties of the cell wall and cell membrane of several mycobacteria and of Leprosy Derived Corynebacteria are investigated by freeze-etching and freeze-fracture. In all cases the freeze-fracture split the cell wall in two asymmetric halves. The cell wall fracture faces of the mycobacteria are characterized by a filamentous network which vary with respect to the amount and complexity among microorganism of the same species and even more of different species. In LDC the structure organization of the cell wall and cell membrane differs from that of mycobacteria. The most stricking difference is the presence on the fracture faces of the LDC cell wall of different classes of particulated entities of yet unknown nature. In the mycobacteria and LDC the periseptal annuli likely provide a potential frame for cell envelope and cell membrane assembly.
Animals↗
Investigation about the surface structure of some viruses by the freeze-etching and freeze-drying methods.
An electronmicroscopic study of a suspension of Venezuelan equine encephalomyelitis (VEE) virus by freeze-drying and freeze-etching methods showed that glycoprotein peplomers are located on the surface of the lipoprotein shell. These peplomers are trimeric in shape and form a regular icosahedral surface lattice corresponding to T = 4. The modes of glycoproteins clustering for the two clones of VEE are different. Venezuelan equine encephalomyelitis (VEE) virus belongs to the alpha-viruses of the toga-virus family. Virions of VEE virus have the icosahedral nucleocapsid surrounded by a lipoprotein envelope (peplos) and contain single-stranded RNA. During budding, alpha-virus nucleocapsid acquires a host cell lipid membrane in which virus specific glycoproteins are built in (Bonsdorff et al. 1975, Harrison et al. 1974). Earlier the surface peplomer clustering was investigated only for the sindbis virus (Bonsdorff et al. 1975, Bonsdorff et al. 1978).
Enterovirus↗
Structural features of mesosomes (chondrioids) of Bacillu subtilis after freeze-etching.
Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.
Bacillus subtilis↗
Envelope of mouse mammary tumor virus studied by freeze-etching and freeze-fracture techniques.
As part of a study of the cell surface changes associated with the production of murine mammary tumor virus, the structure of the envelope of this virus has been examined by using freeze-fracture techniques. Both fracture and deep-etch surfaces were examined. The fracture faces contain 10-nm spheres comparable to those observed on fractured plasma membranes, although fewer in number. Surfaces exposed by etching possess a highly regular hexagonal array of pits 25 nm apart. By examining freeze-fracture and freeze-etch preparations of virus with ferritin covalently bound to its surface, it has been determined that the surface exposed by etching is the outer surface of the virus. The pitted exterior surface of the mammary tumor virus appears to be a unique surface structure.
Dimethyl Sulfoxide↗
Fine structure of ependymal cells in the median eminence of the frog and mouse revealed by freeze-etching.
Freeze-etched preparations of the ventricular surfaces of ependymal cells clearly reveal the presence of pinocytotic vesicles opening into the third ventricle and large vacuoles formed by broad cell projections. The density of the vesicular openings is approximately 20 per micron2. The ependymal cells in the median eminence of the frog are adjoined by tight junctions comprised of five to eight interconnected junctional strands, whereas near the median eminence in the mouse only one to two such strands form the tight junction of the ependymal cells. Gap junctions between the adjacent ependymal cells are detected near the median eminence in the mouse but not in the frog.
Animals↗
Ultrastructure of free-living and nitrogen-fixing forms of Rhizobium meliloti as revealed by freeze-etching.
Freeze-etching of Rhizobium meliloti provided considerable insight into the ultrastructure of this bacterium and into the changes accompanying the transformation from the free-living rod forms to the nitrogen-fixing bacteroid forms. In the small rods, one cleavage plane was revealed at the level of the cell wall and a second at the level of the plasma membrane. Very little structure was evident at the cell wall level, but distinctly different convex and concave fracture faces were exposed at the cell membrane cleavage plane. During the transformation into the bacteroidal state the wall decreased in thickness, became less rigid, and developed a particulate surface. In addition, changes in particle density were observed in the plasma membrane. The fine structure of the plant membranes, the infection threads, and the arrangement of the bacteroids within the plant cells also were revealed.
Cell Membrane↗
Preparation of coenocytes for freeze-etching.
Successful freeze-etching of a coenocyte has been accomplished with glutaraldehyde stabilization followed by infiltration with cryoprotectant. Hyphae of the coenocytic water mold Achlya were stabilized with 5% glutaraldehyde in phosphate buffer. Gradual infiltration by dropwise addition of the cryoprotectant (25% glycerol, 10% ethylene glycol, distilled water, v/v) is accomplished over a period of 8-10 hr on a shaker. Subsequent freeze-etching is carried out by standard procedures.
Cell Membrane↗
Morphology and morphogenesis of Sindbis virus as seen with freeze-etching techniques.
Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.
Animals↗
Quantification of particle sizes with metal replication under standard freeze-etching conditions: a gold ball standard for calibrating shadow widths was used to measure freeze-etched globular proteins.
The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45 degree angle on an approximately -100 degrees C surface in a 10(-6) torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 +/- 0.25 nm, 2.0 +/- 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The "geometric assumption" that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45 degree and 20 degree angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45 degree angle with current high resolution conditions at a substrate temperature of -185 degrees C, and the results of these replicas were compared to the results reported here at approximately -100 degrees C.
Calibration↗
Demonstration of the fuzzy surface coat of rat intestinal microvilli by freeze-etching.
In freeze-etch replicas of epithelial cells of rat rectum, the fuzzy surface coat is composed of discrete filaments which are aligned in parallel with each other and with the long axes of the microvilli.
Animals↗