Chemical cryoprotection for structural studies

With the ascendency of techniques for ultrarapid cooling and the successful control of ice crystal damage by purely physical means, it has become necessary to make a case for the continued use of chemical antifreeze agents in any circumstances. These circumstances include the need to explore tissues deeper than those superficial layers, whose morphology can be preserved by ultrarapid cooling, the avoidance of superficial areas of damage inflicted by dissection and tissue slicing, and situations where the growth of ice crystals must be controlled throughout the specimen, as for example for the cutting of frozen sections. The control of ice crystal damage would ideally be by the avoidance of any freezing at all but in practice can be seen in terms of the control of ice crystal size, which in turn depends on the density of nuclei that develop in the specimen and the rate of accretion of water molecules to the crystallites that form on these nuclei. Chemical antifreeze agents act in a variety of ways to increase the density of nuclei and/or to reduce the rate of growth of ice crystals: by promoting nucleation, by enhancing subcooling and by increasing the viscosity of the extracellular medium. In this way, large numbers of small ice crystals are produced. The different types of cryofixative agent each have their own advocates, advantages, special applications and drawbacks and these must be taken into account when considering the range of methods available for the analysis of cryofixed material.     

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 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.     

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.     

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.     

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.     

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

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

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.     

Comparing different preparation methods to study human fibrin fibers and platelets using TEM

For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl(2) showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets.     

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.     

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.     

Cryopreparation of mammalian tissue for X-ray microanalysis in STEM

A cryopreparation technique for studies of ultrastructure and distribution of diffusible elements in biological tissue is described. Electron microscopical contrast and characteristic X-ray spectra are found to be poor in completely frozen-hydrated ultrathin cryosections of fresh chemically untreated tissue. Both STEM contrast and detection of characteristic X-rays are enhanced by careful freeze-drying in the microscope. Although the ultrastructure is affected by ice crystals, intracellular compartments can be identified by STEM without staining and studied by X-ray microanalysis.     

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 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.     

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.     

Growth of ice crystals in frozen specimens

The formation of ice crystals, which might be the possible artefact in cryo-techniques for electron microscopy, was examined during the rewarming process of rapidly frozen erythrocytes. Intracellular ice formation, which is usually found in cells suspended in saline by rapid freezing, was inhibited by the addition of 30% glycerol. When such glycerinated cells, having no ice crystals at liquid nitrogen temperature, were rewarmed to higher temperatures above ? 80° C, recrystallization of ice occurred. Ice particles became visible within the cells even at ? 80°C and grew larger with a temperature rise. From the results obtained in the morphological and physiological investigations, it also became evident that the recrystallization of ice appeared prior to the increase in haemolysis during the rewarming process.     

Cryo-ultramicrotomy and myofibrillar fine structure: a review.

In the past, the techniques of electron microscopy and X-ray diffraction have both been very informative about the ultrastructure of the muscle myofibril But X-ray diffraction patterns are difficult to interpret unambiguously and until now specimen preservation in plastic embedded muscle has been sufficiently poor to make it difficult to use electron micrographs of muscle as a means of interpreting the available X-ray diffraction evidence. The possibility of using ultrathin sections of frozen muscle, in which the disruptive steps of chemical dehydration and plastic embedding can be avoided, promises to help to bridge the information gap between present X-ray and electron microscope results. For this reason we here review the application of the cryosectioning technique to muscle, we assess the technique in terms of the improvements in preservation which have so far been obtained and which might be expected and we discuss some of the many potential advantages and uses of this technique for studies of muscle ultrastructure and function. It is concluded that this technique should be developed vigorously since it promises to play a very important role in muscle research in the future.     

Cryo-ultramicrotomy and myofibrillar fine structure: a review

In the past, the techniques of electron microscopy and X-ray diffraction have both been very informative about the ultrastructure of the muscle myofibril. But X-ray diffraction patterns are difficult to interpret unambiguously and until now specimen preservation in plastic embedded muscle has been sufficiently poor to make it difficult to use electron micrographs of muscle as a means of interpreting the available X-ray diffraction evidence. The possibility of using ultrathin sections of frozen muscle, in which the disruptive steps of chemical dehydration and plastic embedding can be avoided, promises to help to bridge the information gap between present X-ray and electron microscope results. For this reason we here review the application of the cryosectioning technique to muscle, we assess the technique in terms of the improvements in preservation which have so far been obtained and which might be expected and we discuss some of the many potential advantages and uses of this technique for studies of muscle ultrastructure and function. It is concluded that this technique should be developed vigorously since it promises to play a very important role in muscle research in the future.     

Thermal and radiation damage to frozen hydrated specimens

Although low temperature fixation seems to be superior to chemical fixation with respect to structure preservation, complex interactions between the electron beam and frozen hydrated specimens can limit its applications. A review is given of the present knowledge of beam heating and radiation damage with emphasis on recent developments in the field. Beam heating is found not to be a major limitation to cold stage microscopy. But radiolysis of ice and the formation of free radicals that subsequently attack organic matter in contact with ice can severely alter the specimen structure before a useful image is recorded. Use of low dose techniques may help in some cases, but the problem cannot be overcome in X-ray microanalysis of very small regions, when high beam doses must be used. The use of liquid helium stages operating near 4 K may reduce damage also in frozen hydrated specimens.     

High-pressure freezing of plant cells cultured in cellulose microcapillaries

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