Towards more precise definition of conditions for satisfactory deep etching

Experiments were carried out to determine whether propane-jet freezing could be as satisfactory as impact freezing in deep etch work. The material used was the intestinal brush border, previously studied by Heuser and coworkers where the 5–6 nm decoration of actin rootlet filaments, and the fine network of filaments linking these rootlets, provide good criteria by which to judge the quality of the preparation, as regards ice crystal growth and surface contamination. Propane jet freezing was indeed found satisfactory provided appropriate conditions were met (viz thin specimen, fracture near the surface). Variable results were obtained until it was realized that with a Balzers freeze-etch unit fitted with a rotating specimen table there is a 10–15 min delay (when specimen temperature is reset) between the time the recording thermocouple shows a given temperature to have been obtained and the time the specimen block actually reaches this temperature. Appropriate allowance must be made for this lag to achieve satisfactory deep etch replicas.     

High-pressure freezing for the preservation of biological structure: Theory and practice

The two main advantages of cryofixation over chemical fixation methods are the simultaneous stabilization of all cellular components and the much faster rate of fixation. The main drawback pertains to the limited depth (<20 μm surface layer) to which samples can be well frozen when freezing is carried out under atmospheric conditions. High-pressure freezing increases the depth close to 0.6 mm to which samples can be frozen without the formation of structurally distorting ice crystals. This review discusses the theory of high-pressure freezing, the design of the first commercial high-pressure freezing apparatus (the Balzers HPM 010), the operation of this instrument, the quality of freezing, and novel structural observations made on high-pressure-frozen cells and tissues.     

Application of freeze-etch replication for the observation of the cell-extracellular matrix interface of cultured cells

In order to investigate the ultrastructural three-dimensional relationship between extracellular matrices (ECM) and the plasma membranes of cultured cells, a freeze-etch replica method was devised. Bovine corneal endothelial cells were cultured on a Collodion film which covered a hole punched in a plastic coverslip, and were quickly frozen with a slammer with their basal surface facing a liquid nitrogen-cooled copper block. The cells were placed upside-down in a Balzers freeze-fracture machine and freeze-etched, and then platinum-carbon replicas were obtained. The structure of the ECM-plasma membrane interface was observed successfully and so this technique provides a new approach for investigating the ECM-plasma membrane (matrix-receptor) relationship.     

A freeze-etch electron microscopic study of liquid propane jet-frozen human erythrocyte membranes

Freeze-etch electron micrographs of haemolysing erythrocytes and isolated erythrocyte membranes frozen using a liquid propane jet-freezer reveal fracture faces very different from those seen after conventional freezing by dipping the specimens into partly solidified Freon 22. Instead of the rather smooth extracellular fracture faces found after conventional specimen freezing, extracellular fracture faces exhibiting large amounts of fibre-like structures are seen after liquid propane jet-freezing of these specimens. No such structures were found in normal red blood cells. When isolated erythrocyte membranes are frozen under conditions favouring spectrin-actin release, freeze-etch micrographs reveal an apparent continuity between the fibrelike structures on the extracellular fracture face and the long fibre-like structures which extend from the protoplasmic surface of the erythrocyte membrane. These results suggest that liquid propane jet-freezing is capable of revealing a structural difference between the membrane of haemolysing and nonhaemolysed red blood cells, and that this difference is related to the fibrous, peripheral proteins of the membrane.     

The design and use of a simple device for rapid quench-freezing of biological samples

The detailed design of a simple device for rapid quench-freezing of biological samples under reproducible conditions is presented. With spring-augmented descent, sample immersion velocity of 10 m s^?1 into a cryogenic liquid is achieved. Biological samples, loaded in Balzers planchets, Denton holders, or a newly designed ‘titanium envelope’, are suitable for rapid-freezing with this device. Using 4 μm titanium foil, light weight (1 mg) streamlined holders can easily be made to enclose cell suspensions or tissue samples. The foil envelope is designed for efficient heat dissipation while protecting the sample from possible impact or flow distortions occurring from spring-augmented immersion. Human erythrocytes, quench-frozen in the titanium envelope, were prepared for electron microscopy by the freeze-substitution technique. Two opposing 25–30 μm surface zones were frozen in the apparent absence of ice. The extended depth of cryofixation is attributed to the advantages of thin foil in the titanium envelope design and the use of rapid-immersion technique.     

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

We describe a method for high‐pressure freezing and rapid freeze‐substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed ‘face to face’ without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice‐crystal artefact. Freeze‐substitution at −90°C for 60 min in acetone containing 2% uranyl acetate, followed by warming to −50°C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.     

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog ( Rana pipiens) retina was sliced at a thickness of 150 or 350 μm, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO₄ and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-μm slices or 15% low-MW dextran for 350-μm slices) were required for adequate freezing.
The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-μm slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers.
This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.     

Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

Freeze-fracturing of monolayers (capillary layers) of cell, membranes and viruses: some technical considerations.

A novel hinged device for freeze-fracturing of cell monolayer in the Balzers freeze-etch unit is described. It is economical on biological material and enables oriented adsorption of sheet-like membrane fragments. For freeze-fracturing 'by hand' a monolayer is formed on a positively charged piecie of mica (with polylysine) and this is covered with another piece of mica, thin brass plate of filter paper. Such a sandwich is frozen in liquid nitrogen and fractured by means of forceps. Several modifications of this technique as well as practical examples are described. Among possible application are: negative staining of intramembranous protein particles; chemical or physical analyses of single membrane leaflets; identification of protein complexes by immunoelectron microscopy, etc.     

High-pressure freezing causes structural alterations in phospholipid model membranes

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoylphosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine (1,2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine) have been analysed by freeze-fracture electron microscopy. The liposomes were frozen either by plunging into liquid propane or by HPF. The characteristic macroripple-phase of the two-component liposome system is drastically changed in its morphology when frozen under high-pressure conditions. The influence of ethanol which acts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer membrane have been observed. We assume that the modification of the binary system is due to a pressure-induced relaxation of a stressed and unstable lipid molecule packing configuration. HPF was performed with a newly designed sample holder for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPM010. The sandwich construction turned out to be superior to the original holder system with regard to freeze-fracturing of fluid samples. By inserting a spacer between the supports samples with a thickness of 20–100 μm can be high-pressure frozen. The sandwich holder is provided with a thermocouple to monitor cooling rates and allows exact sample temperature control. Despite a two-fold mass reduction compared to the original holder no HPF cooling rate improvement has been achieved (4000 °C s⁻¹). We conclude that the cooling process in high-pressure freezing is determined mainly by cryogen velocity.     

Structure and function of frozen cells: freezing patterns and post-thaw survival.

Freezing patterns and post-thaw survival of cells varies with different cooling rates. The optimal cooling rates, indicating the highest percentage survival, were different in yeast and red blood cells. A difference of freezing patterns was also noticed in preparations frozen above and below the optimal cooling rate for each cell, namely, cell shrinkage at lower rates and intracellular ice formation at higher rates which showed similar trends in both the cells, even though there was some shifting of the optimum. Ultra-rapid freezing and addition of cryoprotectants are useful ways to minimize ice crystal formation and to cause such ice formations to approach the vitreous state. Ice crystals are hardly detectable in yeast cells as well as in erythrocytes, when these cells are frozen ultra-rapidly in the presence of cryoprotective agents in moderate concentration.     

Structural changes in samples cryofixed by contact with a cold metal block

A common method of cryofixation is to bring a specimen rapidly in contact with a cold metal block. It is usually thought that during this process the surface of the specimen suffers little distortion since it freezes rapidly. Whether this is likely depends on the rate at which samples freeze compared with the speed at which the sample hits the cold block. There is some discrepancy between the published experimentally and theoretically determined freezing rates. As a contribution to this debate the distortion in cryofixed, freeze-substituted, striated muscle fibres has been investigated. In transverse sections, compression can be detected by deviations of the filament lattice from the hexagonal and used to estimate the time of freezing. Some specimens were frozen using a Gatan Cryosnapper, which freezes by catching the specimen between two nitrogen-cooled copper jaws. In addition, the speed with which the jaws close has also been determined. The results suggest that freezing of the well-preserved areas occurs in substantially less than 1 ms. This conclusion is supported by results obtained using metal-mirror apparatus in which the cushioned specimen was dropped onto a nitrogen- or helium-cooled copper block. All the specimens frozen against a cold block have a flat edge whereas muscle fibres are round. At the very edge there is evidence of structural damage as well as the more general lattice distortion.     

Freezing in sealed capillaries for preparation of frozen hydratedsections

We have investigated the freezing of specimens in a confined volume for preparation of vitreous samples for cryosectioning. With 15% dextran as a cryoprotectant, a sample sealed in a copper tube begins to freeze into crystalline ice when plunged into liquid ethane. Crystallization rapidly causes an increase in the pressure to the point that much of the sample freezes in a vitreous state. We used synchrotron X‐ray diffraction of samples frozen with various amounts of dextran to characterize the ice phases and crystal orientation, providing insights on the freezing process. We have characterized cryosections obtained from these samples to explore the optimum amount of cryoprotectant. Images of cryosectioned bacteria frozen with various levels of cryoprotectant illustrate effects of cryoprotectant concentration.     

The use of low temperature X-ray diffraction to evaluate freezing methods used in freeze-fracture electron microscopy

Two methods of freezing samples for freeze-fracture electron microscopy have been compared using X-ray diffraction and freeze-fracture results from lipid-water model systems. Perturbations of the molecular organization of hydrocarbon chains and the extent of ice crystal formation have been evaluated for lamellar phases of egg lecithin containing 16% water and egg lecithin-phosphatidylinositol containing 55% water, both freeze quenched in liquid Freon-22 near its melting temperature (113 K). Very thin samples sandwiched between copper sheets separated by an electron microscope grid show much less freezing induced structural rearrangement than small smaples contained on conventional Balzers-type gold planchettes. These results show that the rate of freezing in the very thin preparations is greater than in the conventional ones, which is probably due in part to the improved dissipation of heat from a poorly conductive sample through highly conductive copper sheets.     

Ice crystal damage in frozen thin sections: freezing effects and their restoration

Thin sections of unfixed kidney, fast frozen without cryoprotectants, were fixed in osmium tetroxide vapour directly after freeze drying or after 30 min in a moist atmosphere. Dry sections fixed in vapour showed ice crystal damage characteristic for the freezing procedure. This was demonstrated with freeze fracture replicas from the same preparation. Ice crystal holes were obscured in serial sections which were freeze dried and allowed to rehydrate in a moist atmosphere. The same ultrastructural appearance was observed in frozen sections brought to room temperature immediately after cutting. Frozen thin sections from unfixed tissue, if freeze dried, are very sensitive to atmospheric conditions and need some form of stabilization (e.g. osmium vapour fixation, sealing with an evaporated carbon film) before electron microscope images can be interpreted as representative for the frozen state. Restoration of ice crystal damage can occur by melting frozen sections or by rehydration of freeze dried frozen sections. Restoration phenomena will impair studies aimed at the localization of diffusible substances by autoradiography or X-ray microanalysis.     

Pre- and post-thaw assessment of intracellular ice formation

Intracellular ice formation (IIF) refers to the formation of ice crystals within cells during rapid freezing. To develop an understanding of the means by which intracellular ice forms and the mechanisms by which it damages cells and tissues requires techniques that combine real‐time assessment of ice nucleation and ice crystal growth with detailed assessments of cell structure and function. Intracellular ice formation has been detected in live samples using light scattering, freeze substitution and fluorescent detection. In this study we develop a method to correlate IIF with post‐thaw structural analyses by combining low temperature microscopy and freeze substitution. V79‐4 hamster fibroblasts were frozen on a low temperature microscope at various temperatures, IIF was visualized using the nucleic acid‐specific fluorophore SYTO 13?, then the samples were fixed (10% formaldehyde, 85% ethanol, 5% acetic acid) while still frozen. The monolayers were then thawed and stained with routine histological stains haematoxylin and eosin and assessed. Fixation allowed for the post‐thaw assessment of IIF and for subsequent histological processing to examine in detail the structural consequences of IIF. The post‐thaw identification of cells that form intracellular ice during freezing is a significant improvement to current methods used in low temperature biology.     

A simple method for quick-freezing

In conventional freeze-fracture replicas produced from tissue cryoprotected with glycerol, the hydrophobic inner surfaces of membranes are revealed, but hydrophillic structures are obscured in the surrounding ice. Quick-freezing of tissue obviates the need for glycerol, which prevents the removal of this ice by etching or freeze-drying, but the major problem in freezing without glycerol cryoprotection is ice crystal formation. We describe here a simple method for quick-freezing tissue, in the absence of glycerol, on a nitrogen-cooled copper block with a hand-held specimen holder. This method freezes samples well enough to preserve molecular detail that can be revealed by subsequent etching. We show some examples of the quality of this freezing with respect to the visualization of molecular detail in isolated protein molecules such as ferritin and catalase. Furthermore, we show examples of in situ cellular structures that are revealed by this method, and we compare the structure seen in these replicas with structures preserved by quick-freezing at liquid helium temperatures.     

Preparation of holey carbon films suitable for cryo-electron microscopy

We present a method for the quick and reliable production of carbon films perforated with 5–10-μm holes, suitable for supporting suspensions that are to be rapidly frozen and imaged, using cryo-electron microscopy, in the frozen-hydrated state. The holes support biological specimens that are often embedded in 50–200 nm of vitreous ice, which is optimal for image contrast and preservation of large macromolecular complexes.     

High-pressure freezing of epithelial cells on sapphire coverslips

Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage‐free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing. In a novel approach, we tested the suitability of sapphire discs as cell substrata for high‐pressure freezing. Frozen samples on sapphire were subjected to freeze‐substitution while in the same flat sample holders as used for high‐pressure freezing. We obtained cells that displayed an excellent preservation of fine structure. Because sapphire is a tissue culture substratum suitable for light microscopy, its use in combination with high‐pressure freezing could become a powerful tool in correlative studies of cell dynamics at light and electron microscopic levels.