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.     

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.     

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.     

Metastable water at subzero temperatures

The origins of physical and chemical metastability in aqueous systems are discussed with particular reference to supersaturated solutions, i.e. those where eutectic phase separation does not normally occur. Such solutions are characterized by high viscosities, and under conditions of rapid cooling to the glass temperature, freezing can be partly or completely inhibited. In such systems water acts as a plasticizer to the solute matrix but is not bound, in the correct sense of the word. At a glass temperature of 228 K, diffusion and evaporation rates of water from the amorphous solid are of the order of 1 nm/day, compared to 10⁴ nm/s for the sublimation of ice at same temperature, despite the fact that ice has a substantially lower vapour 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.     

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.     

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.     

On crystal size and cooling rate

A theoretical model is proposed which is used to derive a quantitative relationship between the critical cooling rate and average crystal size at any location within a biological specimen of given shape subject to rapid freezing. The model is applicable to the slamming, plunging or spraying methods of cryofixation provided the ice crystal size is at least 5 times greater than the size of the critical nucleus. Complete vitrification of pure water or aqueous solutions is shown to take place at cooling rates in excess of about 3 × 10⁶ K/s.     

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Simple procedures for evaluating the cryofixation of biological samples

Ultra-rapid cooling of biological material can be achieved in the absence of cryoprotectants by using thin samples. Three methods now employed to prepare thin samples for freeze-fracture electron microscopy are compared: contacting the sample against a liquid helium-cooled copper surface (Heuser et al., 1979), spraying the sample with a jet of propane (Mueller et al., 1980), and plunging a streamlined copper ‘sandwich’ into liquid propane (Costello, 1980). In the first method a thin surface layer of the sample is ultra-rapidly cooled while in the other methods the entire sample sandwiched between sheets of conducting metals is cooled. The morphology of fracture-faces of dilauryllecithin-water systems is used to evaluate the effectiveness of cooling methods. At optimum cooling rates the initial disordered arrangement of lipid in the lamellar (L_α) phase is preserved, giving smooth fracture faces. At slower cooling rates a worm-like texture appears which signals the formation of molecular ordering characteristic of the P_β, phase. All three methods are capable of cooling these lipid-water phases as well as other more dilute aqueous suspensions without evidence of ice crystal growth or damage. Measurement of cooling rates employing miniature thermocouples embedded in samples indicates that rates for all three methods are in excess of 10,000 K/s. The propane jet (32 times 10³ K/s, slope at 273 K) exposes the sample to coolant more rapidly than the sandwich plunging method (10 times 10³ K/s, slope at 273 K) and therefore produces slightly higher cooling rates for samples of equivalent mass and thickness. Each method has its advantages. The contact method is well suited for tissues; the sandwich method is simple and inexpensive; the jet method can potentially produce the highest cooling rates. The last two methods yield complementary replicas.     

Optimum conditions for cryoquenching of small tissue blocks in liquid coolants

Three approaches were taken with the aim of defining the optimum conditions for rapid cryopreservation in liquid quenchants. In a theoretical approach, two mathematical models were used. The first is of value in defining the absolute maximum rates of cooling which could be achieved at various depths in the tissues. The second highlights the poor thermal properties of liquid coolants and therefore emphasizes the essential requirement for vigorous quenchant mixing and rapid specimen entry. Experimental work with thermocouples showed that fastest cooling rates occur at the leading edge of the object entering coolant. Of five liquid quenchants investigated, cooling rates were in the order, propane> Freon 22> Freon 12> liquid nitrogen slush> liquid nitrogen. Other considerations, however, may affect the choice of quenchant. For a given quenchant, cooling rate is maximal near the equilibrium freezing point. The consequences of quenching in the presence of thermal gradients either within the coolant or in the gas layer above it are shown. Cooling rate was found to be approximately proportional to entry velocity at least up to ～2 m s^?1 in our system. Stereological analysis of rapidly quenched, freeze-substituted tissue samples, of geometry which imposed an approximately unidirectional heat flow, revealed four zones: (i) a narrow surface layer (～10 μm) of low image contrast and apparent absence of ice crystals; (ii) a zone of enhanced contrast with ice crystals whose size increased rapidly with depth from the surface (the ‘slope’); (iii) a sharply defined zone (the ‘ridge’) of maximum ice crystal size beyond which there is (iv) an extensive ‘plateau’ with smaller ice crystals and no marked increase in size with depth. The ‘ridge’ of maximal ice-crystal damage was consistently found but varied considerably in depth from the surface (～25–120 μm) between samples. The existence of the deeper plateau region of relatively uniform ice-crystal-size may be of significance in X-ray microanalytical studies of physiological processes at some depth from the sample surface. In terms of our present understanding of the quenching process, the conditions for optimal cryofixation of small tissue samples are listed.     

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.     

Structural and functional aspects of biological freezing techniques.

The cooling procedures used to prepare samples for ultrastructural examination at low temperatures often differ markedly from those used to recover optimal function of cells on thawing. The implications of these differences are reviewed. Damage and alteration to the structure and function of the cells may be caused by the high concentrations of cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) often added to reduce ice crystal artefacts. Under the rapid cooling conditions commonly employed for structural studies, these additives are not cryoprotective; low rates of cooling are necessary for them to be effective. Rapidly cooled cells that contain intracellular ice are only injured during rewarming so their structure may be as yet unaltered by any damaging effects at low temperatures. Most cells able to recover on thawing are grossly shrunken at low temperatures but since they are potentially functional they are of interest structurally. These cryobiological principles are illustrated with freeze-fracture, freeze substitution and functional assays. The cell types chosen were Chlorella sp. and mammalian tissue culture cells.     

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.     

高温合金蜂窝芯冰固持低损伤加工技术研究

针对金属蜂窝芯存在的薄壁多孔、各向异性、面内弱刚性、径向强度小等加工难题,提出了高温合金蜂窝芯冰固持低损伤加工方法.分析了金属蜂窝芯冰固持装夹原理,验证了工艺系统的适应性和可靠性.开展了蜂窝芯冰固持超低温冷却加工的单因素试验,阐明了蜂窝芯加工缺陷形成规律.试验结果表明:在金属蜂窝芯加工中引入冰固持超低温冷却的装夹和加工...     

氯化钠冰浆系统的试验研究

对刮片式冰浆生成器进行试验研究,探讨不同浓度下的氯化钠溶液结晶的试验过程与现象。试验结果表明:对浓度为3%⁵%的氯化钠溶液制取冰浆,能效系数最高达到1.9。纯净水和氯化钠溶液在试验过程中,存在一定的过冷度下才开始结晶,并且随着浓度的增加冰点温度呈线性减小,与理论相比,由于氯化钠溶液在低温下容易发生析晶,试验得到的溶液结晶点温度比理论的温度要高一些。     

Automated sample exchange and tracking system for neutron research at cryogenic temperatures

An automated system for sample exchange and tracking in a cryogenic environment and under remote computer control was developed. Up to 24 sample "cans" per cycle can be inserted and retrieved in a programed sequence. A video camera acquires a unique identification marked on the sample can to provide a record of the sequence. All operations are coordinated via a LABVIEW program that can be operated locally or over a network. The samples are contained in vanadium cans of 6-10 mm in diameter and equipped with a hermetically sealed lid that interfaces with the sample handler. The system uses a closed-cycle refrigerator (CCR) for cooling. The sample was delivered to a precooling location that was at a temperature of approximately 25 K, after several minutes, it was moved onto a "landing pad" at approximately 10 K that locates the sample in the probe beam. After the sample was released onto the landing pad, the sample handler was retracted. Reading the sample identification and the exchange operation takes approximately 2 min. The time to cool the sample from ambient temperature to approximately 10 K was approximately 7 min including precooling time. The cooling time increases to approximately 12 min if precooling is not used. Small differences in cooling rate were observed between sample materials and for different sample can sizes. Filling the sample well and the sample can with low pressure helium is essential to provide heat transfer and to achieve useful cooling rates. A resistive heating coil can be used to offset the refrigeration so that temperatures up to approximately 350 K can be accessed and controlled using a proportional-integral-derivative control loop. The time for the landing pad to cool to approximately 10 K after it has been heated to approximately 240 K was approximately 20 min.     

An improved transfer module and variable temperature control for a simple commercial cooling holder

A new cold stage transfer module was designed for the commercially available cooling holder of the JEOL JEM 100CX electron microscope. In the new CSTM the entire loading of the specimen is carried out under liquid nitrogen. This gives a frost-free transfer during which the temperature of the sample does not exceed 120 K. Straightforward modifications to the commercial cooling holder permit continuous selection of specimen temperature between 100 and 450 K. The sample can be heated or cooled at rates of up to 7 K/s. These modifications do not impair the resolution of the holder which is better than 1.5 nm. This work illustrates a relatively simple way of modifying a commercial cooling holder into a true cold stage system.     

渔船余热朗肯-朗肯制冰系统研究

为有效利用渔船烟气和冷却水的余热,本文采用有机朗肯-朗肯系统进行制冰,建立了系统的热力学模型,研究了系统的性能参数和影响因素,评价了单位质量热水以及单位功率热源的制冰能力。结果表明:余热温度和冷凝温度对系统性能有重要影响,而冷凝器的过冷温度对系统性能的影响很小。在热源温度为100℃,冷凝温度为40℃时,每吨热水的制冰量为86.4kg/t,单位功率余热每小时的制冰量为2.27kg/(kW.h),论证了有机朗肯-朗肯循环系统用于渔船余热制冰的可行性。     

水流外掠单体冰柱融化速率试验研究

建立水流外掠冰柱实验台,分别开展了水流速度(0.02、0.03、0.04、0.05、0.06和0.07m/s)、水流温度(7、10、13、16和19℃)、冰柱直径(60、70、80和105mm)及冰柱初始温度(-12、-8和-5℃)对于冰柱融化速率的影响研究。通过对融化时间的统计和分析,获得以下结论:水流温度对于融化时间的影响呈幂函数规律;水流速度和冰柱直径对于融化时间的影响呈线性规律;冰柱初始温度对于融化时间影响较小。