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.     

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans , could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.     

Amorphous solid water produced by cryosectioning of crystalline ice at 113 K

Amorphous solid (vitreous) water can be obtained by a number of methods, including quick freezing of a very small volume of pure water, low pressure condensation of water vapour on a cold substrate or transformation of hexagonal ice (the ice which is naturally formed) under very high pressure at liquid nitrogen temperature. Larger volumes can be vitrified if cryoprotectant is added or when samples are frozen under high pressure. We show that a sample of 17.5% dextran solution or mouse brain tissue, respectively, frozen under high pressure (200 MPa) into cubic or hexagonal ice can be transformed into vitreous water by the very process of cryosectioning. The vitreous sections obtained by this procedure differ from cryosections obtained from vitreous samples by the irregular aspect of the sections and by small but significant differences in the electron diffraction patterns. For the growing community of cryo‐ultramicrotomists it is important to know that vitrification can occur at the knife edge. A vitreous sample is considered to show the best possible structural preservation. The sort of vitrification described here, however, can lead to bad structural preservation and is therefore considered to be a pitfall. Furthermore, we compare these sections with other forms of amorphous solid water and find it similar to high density amorphous water produced at very high pressures (about 1 GPa) from hexagonal ice and annealed close to its transformation temperature at 117 K.     

Vitrification depth can be increased more than 10-fold by high-pressure freezing

Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2 kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation. Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high-pressure vitrification depth in a 15–20% sugar solution averages 200 μm. Fully vitrified specimens up to 700 μm in thickness are obtained. When crystalline water is observed it is frequently in the form of high-density ice II, III or IX. These results are probably also relevant for typical biological specimens. The advantage of high-pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.     

Electron cryomicroscopy of frozen-hydrated biological specimens: analysis of freezing artifacts by X-ray cryocrystallography.

Freezing artifacts have been evaluated by X-ray cryocrystallography on pellets of two-dimensional membrane protein crystals: purple membrane and maltoporin. The comparison of the X-ray patterns recorded when the specimens are maintained at room temperature to those obtained when the specimens are maintained at about -160 degrees C shows that (i) membrane proteins have a positive thermal dilatation coefficient: the protein crystal lattice shrinks upon cooling; (ii) the asymmetric unit of crystal containing water is changed upon freezing; the relative intensities of the diffraction rings of such crystals are different after freezing. From these results, it can be postulated that freezing may lead to partial dehydration of biological objects. Electron cryomicroscopy visualizes objects which are structurally influenced by the cooling procedure. However, our microscopy study on maltoporin crystals shows that freezing artifacts are negligible in comparison to artifacts associated with conventional techniques such as negative staining.     

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.     

Electron-beam-induced amorphization of ice III or IX obtained by high-pressure freezing

Cryo-electron microscopy of vitrified specimens makes it possible to observe fully hydrated biological samples unimpaired by chemical fixation, staining and dehydration. High-pressure freezing represents important progress since it allows a 10-fold increase in the vitrification depth. High-pressure freezing can also induce the formation of undesirable high-pressure forms of ice. We show that ice III or IX is amorphized under the electron beam at a dose of about 2400 electronsnm⁻² and that the resulting amorphous ice is similar to the vitreous water obtained by high-pressure freezing.     

A fast method to study the secretory activity of neuroendocrine cells at the ultrastructural level

Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.     

A device for the rapid freezing of biological specimens under precisely controlled and reproducible conditions

The construction and preliminary testing of a device is described which can be used to freeze biological specimens in any cryogenic liquid at temperatures down to the nitrogen freezing point (63 K) and which can operate in the pressure range 1.3 kNm^?2 to 1 MNm^?2. Ultra-rapid freezing can be carried out in a subcooled cryogenic liquid either hyperbarically or at atmospheric pressure. Slow freezing rates can be achieved by cooling the specimens in a controlled manner in the vapour phase above the liquid bath.     

Rapid freezing and electron microscopy for the arrest of physiological processes

An apparatus for the rapid freezing of tissue is described, which can be used for the electron microscopy of arrested physiological processes. The material is frozen by bringing it in contact with a silver surface cooled to liquid nitrogen temperature at reduced pressure. The freezing surface is protected from condensation of moisture and gases from the air by a flow of helium gas. The cooling of the specimen during its descent through the cold helium is not large enough to interfere with physiological processes. Freezing occurs very rapidly in the surface but is retarded to about 8 msec at a depth of 10 μm. The apparatus was used to freeze frog muscle during contraction.     

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.     

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.     

Coating sections for electron microscopic autoradiography: a stripping technique using liquid emulsion.

A coating technique for electron microscope autoradiography is described which combines the advantage of forming an emulsion film by a dipping method with the ease of coating sections already on grids. Sections are coated so that a formvar support film separates the section and the emulsion crystals. This intermediate layer of formvar ensures a random distribution of the emulsion crystals. Using light gold sections, Ilford L-4 emulsion and Microdol-X development, the resolution of this technique, as determined by the half distance method, was 150 nm. The additional layer of formvar slightly reduced the image quality with biological samples in the electron microscope. This technique has a minimal loss of resolution and image quality for moderate resolution electron microscope autoradiography.     

A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues

In this paper, we review some published studies using correlative light and electron microscopy methods. We further refined our criteria to include only those studies using live cells for light microscope and where high-pressure freezing was the method of specimen preparation for electron microscopy. High-pressure freezing is especially important for some difficult-to-fix samples, and for optimal preservation of ultrastructure in samples larger than a few micrometres. How the light microscope observations are done is completely sample dependent, but the choice of high-pressure freezer depends on the speed required to capture (freeze) the biological event of interest. For events requiring high time resolution (in the 4–5 s range) the Leica EM PACT2 with rapid transfer system works well. For correlative work on structures of interest that are either non-motile or moving slowly (minutes rather than seconds), any make of high-pressure freezer will work. We also report on some efforts to improve the capabilities of the Leica EM PACT2 rapid transfer system.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

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.     

High-pressure freezing for immunocytochemistry

Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to ≈ 15 μm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystal-artefact-free freezing of samples up to 200 μm in thickness. There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze-substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.     

Comparison of slam-freezing and high-pressure freezing effects on the DNA cholesteric liquid crystalline structure

Using in parallel electron microscopy of ultrathin frozen-hydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.     

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.