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.     

Principles for the construction and operation of a device for rapidly freezing suspensions for cryo-electron microscopy

Details are given concerning the construction and use of a simple device for preparing samples of frozen-hydrated biological material embedded in thin films of vitreous ice. The control of humidity around the sample and rate of freezing are of prime importance to obtain good specimens for cryo-electron microscopy. Symptoms of ill performance of such an apparatus and poor cryo-manipulation are discussed.     

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.     

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.     

Microcarriers for high-pressure freezing and cryosectioning of adherent cells

A method is described employing microcarrier spheres of cross‐linked dextran for obtaining ultra‐ and semithin vitreous sections from high‐pressure frozen anchorage‐dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen‐hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the ‘life cycle’ of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high‐pressure freezing.     

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.     

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.     

Advances in ultrarapid freezing for the preservation of cellular ultrastructure

Most of our current knowledge of cellular ultrastructure is derived from studies of chemically fixed and chemically cryoprotected preparations. In the first part of this review, we document the many artifacts associated with chemical techniques that render them unsuitable for further refinement of our understanding of cellular ultrastructure. The best method currently available for the preservation of cellular ultrastructure is ultrarapid freezing. The second part of this review is a consideration of the physics of ice crystal formation in biological systems, which suggests that ice crystals will be present in any frozen, uncryoprotected specimen. We define an ultrarapidly frozen preparation as one in which the ice crystals are so small as to be invisible at the electron microscopic level. Improvements in the ease of application and reliability of ultrarapid freezing techniques have reached the point that these techniques can be used by anyone requiring the best achievable preservation of cellular ultrastructure. In the third part of this review, we describe and critique the five methods of ultrarapid freezing in current use.     

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.     

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.     

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.     

Cryo-SEM method for the observation of entrapped bubbles and degree of water filling in large wet powder compacts

There are generally two problems associated with cryogenic scanning electron microscopy (cryo-SEM) observations of large wet powder compacts. First, because water cannot be vitrified in such samples, formation of artefacts is unavoidable. Second, large frozen samples are difficult to fracture but also to machine into regular pieces which fit in standard holders, especially if made of hard materials like ceramics. In this article, we first describe a simple method for planning hard cryo-samples and a low-cost technique for cryo-fracture and transfer of large specimens. Subsequently, after applying the entire procedure to green pellets of iron ore produced by balling, we compare the influence of plunge- and unidirectional freezing on large entrapped bubbles throughout the samples as well as the degree of water filling at the outer surface of the pellets. By carefully investigating the presence of artefacts in large areas of the samples and by controlling the orientation of the sample during freezing and preparation, we demonstrate that unidirectional freezing enables the observation of large entrapped bubbles with minimum formation of artefacts, whereas plunge freezing is preferable for the characterization of the degree of water filling at the outer surface of wet powder compacts. The minimum formation of artefacts was due to the high packing density of the iron ore particles in the matrix.     

Computer automated cell size and shape analysis in cryomicroscopy

Computer vision techniques have been developed for quantitative analysis of size and shape changes in cells frozen on a cryomicroscope. The analysis is based on implementation of standard serial edge detection algorithms in conjunction with a shape transform to isolate individual cells in complex scenes which may include adjacent and overlying ice crystals. In the present study the sensitivity of the automated analysis procedure is evaluated for images obtained by various microscope optical systems for progressive degrees of subject blurring by defocusing. Size measurements in calibration trials for freezing latex spheres with extracellular ice in the field of view were least sensitive for bright field images, although the most consistent data was obtained by differential interference contrast microscopy. In all cases phase contrast images produced the least accurate data. An example analysis is presented for the freezing of pancreas β-cells.     

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.     

A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension

We have developed a quick-freezing method, using a copper block cooled with liquid helium or nitrogen, which permits us to freeze muscles without any cryoprotectant at predetermined, precisely measured points in the recorded tension time-course of a single twitch or tetanus. Our aim is to arrest structural intermediates of the cross-bridge cycle for observation in the electron microscope. Chemically stimulated, demembranated muscles as well as electrically stimulated, live muscles can be frozen on the same apparatus. Good freezing of relaxed and contracting muscles has been obtained to a depth of 10–20 μm, with excellent structural preservation after freeze-substitution.     

Study of the conditions necessary for propane-jet freezing of fresh biological tissues without detectable ice formation

The performance of a commercial double-propane-jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze-etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20–30μm thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze-etch microtome within 5–10μm of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.     

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.     

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.     

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.     

Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions

The freezing of biological cell suspensions can be understood in terms of ice formation in the external suspension medium and the cellular reactions to the changing environment. Cryomicroscopy allows a quantitative analysis of both categories of phenomena. Besides freezing stages of appropriate thermal design, the components used for that purpose include a microcomputer (PSI 80) based control system, an image analysis system (Intellect 100) and a spectrophotometer (MPV compact). The investigation of extracellular ice formation is focused on the following effects: The redistribution of solutes in the residual liquid and the resulting concentration profiles are determined photometrically or densitometrically. The transitions between various morphologies of the ice–liquid phase boundary (planar–cellular–dendritic) can be related to interface instability theories. With respect to solute segregation, the studies also involve the formation of bubbles from supersaturated gaseous solutes and freezing potentials resulting from the differential incorporation of cations and anions into the solid phase. The interaction between particles or cells and the advancing ice front is determined from critical interface velocities marking the transition between repulsion and entrapment. The effects of freezing on biological cells are studied mainly with blood cells, especially lymphocytes. The water efflux due to osmotical gradients across the membrane yields volume shrinkage curves which are recorded and analysed from video images for various cooling rates. Beyond a certain threshold cooling rate, intracellular ice starts to form, and different crystallization morphologies can be detected. The intracellular crystallization temperatures depend on cooling and warming rates as well as on the presence of penetrating cryoadditives. A fluorescence viability is used to determine the percentage of damaged cells immediately after thawing.