Heptane and isooctane as embedding fluids for high-pressure freezing of Petunia ovules followed by freeze-substitution

In comparison with other fixation methods, high-pressure freezing and freeze-substitution of Petunia ovules lead to improved ultrastructural preservation of all tissues. Crucial for adequate high-pressure freezing is the absence of air in the specimen sandwich; air has to be replaced by an embedding fluid. Frequently, 1-hexadecene is used for this purpose. Using 1-hexadecene as an embedding fluid resulted in only 5–10% of Petunia ovules being preserved without disturbance of the ultrastructure due to ice-crystal damage. Since 1-hexadecene is not soluble in acetone at − 90 °C, freeze-substitution is hindered when ovules remained completely surrounded by it; this results in recrystallization when the temperature is raised. We tested and compared the suitability of heptane and isooctane as embedding fluids for high-pressure freezing and freeze-substitution, reasoning that because of their low melting points and low relative densities, phase separation during freeze-substitution would result in complete exposure of the ovules to the substitution medium, leading to adequate freeze-substitution. Using either heptane or isooctane as an embedding fluid yielded up to 90% ice-crystal-free ovules. Both compounds, however, have some damaging effects on the outer one or two cell layers of the ovule, but not on the inner tissues.     

High-pressure freezing of tissue obtained by fine-needle biopsy

High-pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze-substitution) of biological tissue up to a thickness of about 200 μm. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine-needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.     

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.     

Freeze-substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K⁺ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used; so far, in the best case, about 67% of the cellular K⁺ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

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.     

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.     

Freeze-substitution of plant tissues with a new medium containing dimethoxypropane*

Meristematic cells from root tips of Dahlia variabilis (L.) and highly vacuolated storage parenchyma cells of Helianthus tuberosus (L.) were fixed by the high-pressure freezing technique and freeze-substituted. A new water-free medium for freeze-substitution was developed to obtain better preservation of the ultrastructure of plant tissues. Adding dimethoxypropane (DMP) to the substitution medium gave two positive results: (1) water could be withdrawn from the samples without an exchange of the medium and without adding drying agents or working in a nitrogen atmosphere, and (2) the ultrastructural preservation of the substituted tissues was better than in a methanol medium and at least as good as in tissues fixed chemically at room temperature.     

Freezing of aqueous specimens: an X-ray diffraction study

The effects on water of two cooling methods, immersion in a liquid cryogen and high-pressure freezing, were studied by X-ray cryodiffraction on different sucrose solutions. The nature of the ice formed by each method depends on both the sucrose concentration and the specimen thickness. In order to compare the two methods, we mainly studied specimens having a thickness of 0.2 mm. Under these conditions, freezing by immersion gives rise to hexagonal (IH), cubic (IC) and amorphous (IV) ices when the sucrose concentration (weight/weight) has a value within the range 0–30%, 30–60%, 60% and higher, respectively. The temperature of the phase transitions IV–IC, IC–IH depends on the sucrose concentration. High-pressure freezing gives rise to two specific forms of ice: an amorphous and a crystalline ice (ice III). Ice III is observed when pure water samples are high-pressure frozen provided that the sample temperature does not rise above −150 °C. Above this temperature, ice III transforms into hexagonal ice. Amorphous ice is formed when the sucrose concentration is higher than 20%. The amorphous ice formed under high pressure has a similar, but not identical, X-ray diffraction pattern to that of amorphous ice formed at atmospheric pressure. While the X-ray diffraction pattern of amorphous ice formed at atmospheric pressure (IV) shows a broad ring at a position corresponding to 0.37 nm, that of high-pressure amorphous ice (IVHP) shows a broader ring, located at 0.35 nm. IVHP presents a phase transition (IVHP–IV) at temperatures that depend on the sucrose concentration. We also observed that some precautions have to be taken in order to minimize the alcohol contamination of high-pressure frozen samples. The ice-phase diagram presented in this paper should be taken into account in all methods dedicated to the structural study of frozen biological specimens.     

Cryofixation of plant tissues without pretreatment

Root tips from Sorghum and Dahlia were frozen without cryoprotection by dipping into nitrogen slush, rapid immersion in liquid propane and by the high-pressure method. Structural preservation of the samples was studied using freeze-fracture (FF) and freeze-substitution (FS) techniques for electron microscopy. It was found that most of the organelles were disrupted by freezing in nitrogen slush and that only the boundary beween the cytoplasm and the vacuole remained visible. If the samples were frozen by rapid immersion in liquid propane, small membraneous organelles, such as dictyosomes, were preserved in peripheral regions of the rhizodermal cells up to 10 μm below the surface of the tissue. Specimens frozen by the high-pressure freezing technique showed good ultrastructural preservation throughout the tissues up to a depth of more than 100 μm.     

High-pressure freezing of plant cells cultured in cellulose microcapillaries

A new microculturing technique for plant cells was used to meet the requirements of high-pressure freezing (HPF). The plant cells were cultured inside cellulose microcapillaries, providing an easy-to-handle method for a real in situ fixation. The high viability of the cells was demonstrated by regenerating shoots from microcalluses cultivated by this method. In general, the freezing quality of the high-pressure frozen samples was excellent across the whole diameter of the capillaries, as shown with ultrathin sectioned cells after freeze-substitution and embedding in Spurr's resin. In comparison with conventional chemically fixed cells, cultured under identical conditions, all membranous compartments and organelles were more turgid and smoother after HPF. The cytoplasm and the matrix of the organelles were more homogeneous and dense. Thus, high-pressure freezing in combination with the microculture method described here appears to preserve the ultrastructure of chemically untreated plant cells close to the native state.     

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells.
Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in a plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40–75 μm from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.     

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21°C)

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl-sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min^?1 or 2 K min^?1 were related to the distribution of the ice phase within the tissue.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution

Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.     

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.     

Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry

Cryo-fixation followed by freeze-substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze-substituted at ?80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well-preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation-resistant and fixation-labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR-75-1 at both light and electron microscope levels.     

Chloroplast ultrastructure in leaves of Urtica dioica L. analyzed after high-pressure freezing and freeze-substitution and compared with conventional fixation followed by room temperature dehydration

In this article, we report on the adaptation of high-pressure freezing and freeze-substitution (HPF-FS) for ultrastructural analysis of leaf tissue with special emphasis on chloroplasts. To replace the gas in the intercellular spaces, a mixture of water and methanol (MeOH) was employed. We compared three different supplements for FS--osmiumtetroxide, uranyl acetate, and safranin--with regard to the preservation of the ultrastructure of chloroplasts and other cellular compartments. The results show that (i) replacement of air within intercellular spaces by 8% (v/v) MeOH has no influence on the ultrastructure of the chloroplasts, (ii) undulation of membranes frequently observed after conventional preparation of specimens does not occur during chemical fixation but during room temperature dehydration, and (iii) uranyl acetate or osmium tetroxide employed during FS are not superior over safranin.     

Vitrification of articular cartilage by high-pressure freezing

For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high-pressure freezing using theoretical and experimental methods. A new high-pressure freezing apparatus (Lei?a EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150-μm-thick tissue samples. Vitrification was proven by subjecting frozen-hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone-dependent attainment of vitrification in 150-μm-thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high-pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.     

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.     

Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.