Intracellular ice formation: cryomicroscopical observation and calorimetric measurement

The formation of ice crystals within biological cells is generally deleterious and results in a severe loss of cellular viability and function. With the aim of circumventing this lethal event, the mechanisms of nucleation and their dependence on governing parameters such as temperature, cooling rate and solute and/or additive concentration, and the correlation with the osmotically induced water transport across the cell membrane were investigated. Quantitative low-temperature light microscopy was used for this purpose as it offers the major advantage of studying the dynamics of the involved processes. To substantiate further the visual observations of the morphological changes associated with intracellular ice formation, supplementary studies by differential scanning calorimetry (DSC) were performed under comparable conditions to measure the quantity of water actually transformed into the crystalline state due to the evolution of latent heat. Human lymphocytes were used as a biological model cell. In particular it could be shown that the twitching type of intracellular ice formation which is evident but difficult to observe under the cryomicroscope can be attributed to a liquid-solid phase change within the cells as determined by DSC. Good agreement was obtained between the results measured by both techniques with respect to the following dependencies of governing parameters: the fraction of cells exhibiting intracellular ice determined as a function of the cooling rate shows a sharp demarcation zone with an increase from 0 to 100% at about the same threshold cooling rate. On the other hand, the temperatures at which intracellular ice forms were found to be only weakly dependent on the cooling rate. With respect to the effect of cryo-additive concentration at a fixed value of the cooling rate, the crystallization temperatures were seen to decrease with concentration. The DSC results may hence be regarded as a validation of the microscopic observations.     

Thermally defined cryomicroscopy and some applications on human leucocytes

A freezing-stage has been developed for use on a standard light-microscope, which can provide reproducible, precisely linear cooling and warming rates in the range from 0·1 to 10,000 K/min. Biological cells in aqueous solutions can be observed during the freeze-thaw cycle; the volume loss due to osmotic efflux of water and the intracellular crystallization of water are detected by video-monitoring. The temperature field generated in the observed samples is comparable to extended cylindrical probes and allows the transfer of cryomicroscopic data to technically used vial geometries. Lymphocytes and granulocytes were observed during freezing using the system described. They were separated and washed, and then frozen on the cold stage of the cryomicroscope at cooling rates ranging from 2 to 500 K/min. Shrinkage of the cells was observed up to 100 K/min and intracellular ice formation could be detected starting at 10 K/min. The results show that human leucocytes show excessive shrinkage up to 36% of their initial volume; the probability of intracellular ice formation exhibits a sharp increase from 10 to 100 K/min where nearly all cells contain ice.     

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.     

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.     

An optical-axis freezing stage for laser-scanning microscopy of broad ice–water interfaces

This article presents a method to view a dynamic ice interface along the axis of ice growth using a laser‐scanning microscope. A deep liquid volume is chilled from below so that ice growth is directed upward toward the microscope objective. The interface is made visible by rejection of fluorescent dye from the solid phase into the liquid. Images of the interface morphology in water with solutes of interest to cryobiology illustrate the imaging capability. These images are processed to quantify the lamellar structure of the ice interface. The optical‐axis cryostage provides advantages over horizontal arrangements because (1) immersion objectives enhance, rather than disturb, the desired thermal gradient, and (2) features in the ice interface are not confined within a narrow capillary tube or microscope slide. This arrangement loses some of the thermal control found in planar freezing stages, and the dynamic, refractive interface presents challenges to confocal microscopy.     

贵金属Au凝固行为的计算机模拟

采用分子动力学方法和量子修正的Sutton-Chen(QSC)多体势,研究了不同冷速下贵金属Au在1700～300K之间的冷却过程,并用径向分布函数、键对分析技术方法对Au在凝固过程中微观结构演变情况,进行了分析研究,结果表明,随着冷速的降低,Au的微观结构从非晶态发展到晶体结构,并且冷速越低,面心立方结构在体系中越占有优势,其结晶的温度也越接近熔点。     

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.     

Crystalline ice as a cryoprotectant: theoretical calculation of cooling speed in capillary tubes

It is generally assumed that vitrification of both cells and the surrounding medium provides the best preservation of ultrastructure of biological material for study by electron microscopy. At the same time it is known that the cell cytoplasm may provide substantial cryoprotection for internal cell structure even when the medium crystallizes. Thus, vitrification of the medium is not essential for good structural preservation. By contrast, a high cooling rate is an essential factor for good cryopreservation because it limits phase separation and movement of cellular components during freezing, thus preserving the native-like state. Here we present calculations of freezing rates that incorporate the effect of medium crystallization, using finite difference methods. We demonstrate that crystallization of the medium in capillary tubes may increase the cooling rate of suspended cells by a factor of 25-300 depending on the distance from the centre. We conclude that crystallization of the medium, for example due to low cryoprotectant content, may actually improve cryopreservation of some samples in a near native state.     

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.     

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.     

Freezing patterns in quench frozen,freeze-dried polyvinylpyrrolidone (PVP)

An SEM investigation of the effects of freezing and freeze-drying on 2 mm diameter columns as well as on 10 mm diameter thick discs of PVP was carried out using standard techniques of quench freezing and cryofracturing followed by freeze-drying. Quench freezing in liquid Freon 12 or liquid propane led to patterns of ice formation that depended on the position in the sample, the orientation of the fracture plane observed relative to the geometry of the sample and the concentration of PVP. Vitrification did not occur. Quench freezing in liquid N₂ resulted in larger ice crystals and the pattern of the crystals was much less predictable.     

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.     

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.     

Natural propane cryogen for frozen-hydrated biological specimens

The properties of natural propane, mixed with 0–4% isopentane, as a cryogen suitable for rapid freezing of this layers of aqueous biological specimen suspensions are discussed. Although natural propane has rather variable properties, its freezing point can be depressed below the temperature of liquid nitrogen by adding a smaller amount of isopentane than is required for depressing the freezing point of pure propane.     

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.     

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.     

An ultrastructural study of cryofractured myocardial cells with special attention to the relationship between mitochondria and sarcoplasmic reticulum

A comparative study of various cryofracturing techniques has been conducted on the mammallian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube-like structures in the scanning electron microscope (SEM). Regular cross-bridging often seen at the Z-band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably. Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometres under the surface. The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol-impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles. Separation of the cell organelles caused by freezing or by critical point drying revealed thread-like structures extending from the mitochondrial surface. Re-examination of SEM-processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron-dense material, here named ‘mito-reticular junctional fibres’, bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specific anchoring function.     

On estimating freezing times during tissue rapid freezing

For the study of morphological changes that are associated with fast physiological processes, it is important to know the times at which the surface regions of specimens are frozen during rapid freezing. A simple physical model has been used to estimate the freezing times and the cooling rates at 10 μm depths in specimens. The calculations indicate that cooling rates in excess of 4 × 10⁴ K s^?1 are associated with freezing times of less than 0.5 ms. Using the same model, experimental measurements of freezing times at much larger depths have been extrapolated to a depth of 10 μm, the times obtained are 0.1-0.6 ms for freezing by rapid immersion in cryogenic liquids, and 0.1 ms or less for freezing on a metal block. It is concluded that the delay time between contact with a cryogenic source and specimen freezing is less than 0.5 ms. The uncertainty in the time of freezing may be larger than this, because of an uncertainty of about ± 0.5 ms in determining the exact time of contact and, for freeze fracture studies, because of an uncertainty of up to 0.5 ms due to imprecision in the depth of fracture. At the same time it is estimated that the time during which freezing takes place may be as high as 250 μs, which can be taken as an upper limit for the resolution time for rapid freezing.     

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.     

干冰升华式天然气水合物孔底冷冻取样器的研制

针对天然气水合物的赋存特点,研制天然气水合物孔底冷冻取样器。取样器是以干冰为冷冻剂,酒精为助冷催化剂和载冷剂的冷冻方式来实现孔底冷冻岩样。介绍了取样器的技术参数、冷冻方式原理、工作原理和结构特点。