Freeze-fracture electron microscopy

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.     

Novel ultrastructural observations of pea (Pisum sativum) root nodule cells by high-pressure freezing and propane-jet freezing techniques

Summary Pea (Pisum sativum) root nodule cells infected by the diazotrophRhizobium leguminosarum have been well characterized by chemical fixation techniques. Propane-jet freezing and high pressure freezing were used in this study to compare rapidly frozen and chemically fixed pea root nodule cells. Cells that had been incubated in 2-(N-morpholino)ethanesulfonic acid buffer and frozen with the propane-jet freezer were better preserved than cells that had been chemically fixed or frozen with the high-pressure freezer. Rapidly frozen infected nodule cells showed that the rough endoplasmic reticulum had a high frequency of associations with the peribacteroid membrane and the infection thread. The peribacteroid space also varied in size depending on the method of preservation; however, it was most reduced in size and devoid of inclusions in the propane-jet frozen tissue. The biological significance of these observations is discussed.Abbreviations HPF high-pressure freezing - MES 2-(N-morpholino)ethanesulfonic acid - PBM peribacteroid membrane - PBS peribacteroid space - PJF propane-jet freezing - RER rough endoplasmic reticulum     

A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella

Chemical fixation protocols provided unsatisfactory preserved material for ultrastructural studies on Jaagiella alpicola Vischer (Chlorophyta). Instead, several methods of rapid freeze fixation followed by freeze substitution were applied. For fast freeze fixation, two methods were tried: plunge immersion freezing in liquid propane using a home-made device, and projection against a copper block cooled by either liquid nitrogen or liquid helium. Each method furnished well fixed material. The quality of the fixed samples was quite similar whether propane or the cryoblock cooled with liquid nitrogen was used. Liquid helium, however, provided superior results. After fixation the samples were cryosubstituted, using acetone or methanol as organic solvent with a chemical fixative added. Acetone gave better results than methanol as a substitution solvent when high temperature embedding was performed. The best cryosubstitution for ultrastructural studies was that in which osmium tetroxide or a mixture of osmium tetroxide and urany acetate was used.     

Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips ofNicotiana andArabidopsis

Summary To circumvent the limitations of chemical fixation (CF) and to gain more reliable structural information about higher plant tissues, we have cryofixed root tips ofNicotiana andArabidopsis by high pressure freezing (HPF). Whereas other freezing techniques preserve tissue to a relatively shallow depth, HPF in conjunction with freeze substitution (FS) resulted in excellent preservation of entire root tips. Compared to CF, in tissue prepared by HPF/FS: (1) the plasmalemma and all internal membranes were much smoother and often coated on the cytoplasmic side by a thin layer of stained material, (2) the plasmalemma was appressed to the cell wall, (3) organelle profiles were rounder, (4) the cytoplasmic, mitochondrial, and amyloplast matrices were denser, (5) vacuoles contained electron dense material, (6) microtubules appeared to be more numerous and straighter, with crossbridges observed between them, (7) cisternae of endoplasmic reticulum (ER) were wider and filled with material, (8) Golgi intercisternal elements were more clearly resolved and were observed between both Golgi vesicles and cisternae, and (9) larger vesicles were associated with Golgi stacks. This study demonstrates that HPF/FS can be used to successfully preserve the ultrastructure of relatively large plant tissues without the use of intracellular cryoprotectants.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FF freeze fracture - FS freeze substitution - HPF high pressure freezing Dedicated to the memory of Professor Oswald Kiermayer     

Low-temperature electron microscopy for the study of polysaccharide ultrastructures in hydrogels. I. Theoretical and technical considerations

The high-pressure freezing (HPF) technique was applied to the cryo-immobilization of alginate gels and the quality of the freezing analyzed on a TEM by comparison of the segregation pattern of samples of decreasing thickness. Dynamic simulations of heat transfer within an idealized slab of pure water surrounded by two walls of aluminium were performed to illustrate the effect of the heat-transfer coefficient by convection on the cooling rate of the sample. Heat-transfer coefficients in liquid nitrogen and liquid propane at ambient pressure were measured using a carefully characterized thermocouple and the values incorporated as parameters in heat-transfer simulations to compare the efficiency of the plunge-freezing technique with the high-pressure freezing technique. Values of the heat-transfer coefficient in liquid nitrogen and liquid propane, calculated between 273 K and 173 K were 670 and 18420 W/m(2)/K, respectively. Based on TEM observations and the results of heat-transfer simulations, the HPF technique was adapted to the cryo-fixation of 50-microm-thick alginate gels. The occurrence of artifacts was rejected because no differences were observed in the pattern of cryo-fixed and freeze-substituted samples of various thickness, with and without ethanol as cryo-protectant. A sample thickness of 50 microm was found to ensure an adequate preservation of structures as small as a few nanometers, as verified by TEM and SEM observations. Finally, DSC measurements on alginate solutions and alginate beads revealed that under the experimental conditions (0-3%), alginate cannot be considered to be an efficient cryo-protectant.     

Recent advances in freeze-fracture electron microscop: the replica immunolabeling technique

Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique samples are frozen fractured and replicated with platinum-carbon as in standard freeze fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.     

Effects of Different Fixatives on Freeze-Fractured Samples of Plant Cells for SEM Observations

Three-dimensional structures of meristematic cells of Allium cepa were studied using freeze-fracture method under the scanning electron microscope. Two fixation procedures were used. The cells were often fractured between eytoplasm and nucleus when the materials were fixed in 1% OsO4 alone before freeze fracture, whereas the nuclei, were frequently fractured if the materials were fixed first in Carnoy's, solution (ethanol: acetic acid=3:l) and then in 1% OsO4 before freeze fracture. The former fixation procedure is suitable for the study of the interior structures of cytoplasm such as cytoskeleton fibres, mitochondria, endoplasmic reticulum and their three-dimensional topography. The latter fixation method is suitable for the study of interior structures of nucleus such as chromatin, nucleoli, nuclear matrix filaments and their 3-dimensional architectures, especially the 3-dimensional structures of chromatin in fibrillar centre of the nucleolus.     

Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues.

Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues.     

Ultrastructure of the zoosporangia of Albugo ipomoeae-panduratae as revealed by conventional chemical fixation and high pressure freezing followed by freeze substitution

Both conventional chemical fixation and high pressure freezing followed by freeze substitution (HPF/FS) were used to prepare zoosporangia of the oomycete Albugo ipomoeae-panduratae inside infected host leaves for study with transmission electron microscopy. Both fixations gave good preservation of ultrastructural details and data from the two sample types were highly complementary. However, HPF/FS gave better overall specimen contrast and superior preservation of microtubules, basal bodies and curved vacuoles closely associated with basal bodies. The basal body-associated vacuoles appear to represent cleavage vesicles involved in zoospore formation. Although HPF/FS did result in the rupture of some vacuoles and the extraction of lipid bodies, these problems did not interfere with our study. Overall zoosporangium morphology was similar to that reported previously for A. candida. Each zoosporangium was multinucleate and contained numerous mitochondria, lipid bodies, a variety of large and small vacoules/vesicles, and conspicuous arrays consisting of parallel strands of rough endoplasmic reticulum. Golgi cisternae and a pair of basal bodies were closely associated with each nucleus.     

Spatial differentiation in photosynthetic and non-photosynthetic membranes of Rhodopseudomonas palustris.

The cytoplasmic membrane and the photosynthetic intracytoplasmic membranes of Rhodopseudomonas palustris are spatially differentiated into regions of extremely high intramembrane-particle density (4,400 to 9,800/micron 2) and areas of lower intramembrane-particle density (2,700 to 5,900/micron 2). The high intramembrane-particle-density areas were always seen in association with photosynthetic membrane stacks. This differentiation was also seen in those areas of the cytoplasmic membrane which adhere to the underlying intracytoplasmic membranes, implying that the cytoplasmic membrane too is differentiated for photosynthesis in these regions. Changes in intramembrane-particle size distribution in response to changes in light intensity during growth were measured. We found that, as light levels were decreased from 8,500 to 100 lx, the average particle diameter in the protoplasmic face of stacked intracytoplasmic and cytoplasmic membranes increased from 8.6 to 10.3 nm. We also observed a distinct periodicity in the sizes of the intramembrane particles found in the stacked regions--7.5, 10.0, 12.5, and 15.0 nm--with the larger-size peaks becoming more pronounced as light intensity decreased. This suggests that, as light levels decrease, subunits of discrete size are being added to a core particle. A comparison of propane jet-frozen cells versus fixed, glycerinated, and then frozen cells indicated that ultrarapid freezing leads to a higher quality of fine-structure preservation than does chemical fixation followed by glycerination and conventional freezing in Freon-12 or propane. The intramembrane particles appeared to be more regular in size, lacking the deformed or jagged appearance displayed in fixed preparations.     

Freeze-fracture autoradiography: feasibility

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane with its chemical and functional characteristics. Within the limits of electron microscope autoradiographic resolution, FARG has the potential to detect the relative distribution of molecules in each half of the membrane and within the plane of the membrane. The use of radioisotopic labels in combination with freezing techniques requires minimal perturbation of the system being studied and may be suitable for the examination of substances which would be extracted or would diffuse during the normal fixation and embedding procedures used in standard electron microscope autoradiography.     

The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.     

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.     

The envelope of intracellular African swine fever virus is composed of a single lipid bilayer

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.     

Cryofixation processing is an excellent method to improve the retention of adrenomedullin antigenicity

We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Ultrastructural visualization of unfixed and unstained whole mounts by high-voltage electron microscopy at low temperatures

A physical procedure for the visualization of cellular fine structures is described as an alternative to chemical preparative techniques. It consists of fixation by fast freezing followed by controlled etching in the cryostage of a million-volt transmission electron microscope. Whole mounts were thus observed under stable conditions with no use of chemical fixatives, solvents, or stains, with no exposure to the atmosphere, and with the improved penetration and resolution in thick specimens that characterize high-voltage electron microscopy. The preservation, contrast, and resolution exhibited by images of preparations obtained by this procedure are discussed.     

DEVELOPMENT AND EVALUATION OF A FREEZE-ETCH,FREEZE-FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM

A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze-etch, freeze-fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen-cooled Freon-22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze-fracture was carried out under standard conditions. Evaluation of replicas from glycerol-imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol-treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol-treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.