Strong evidence against section thawing whilst cutting on the cryo-ultratome

The distribution of ice crystal sizes in frozen thin sections correlates with the size distribution in freeze fracture replicas. In the hypothetical case of melting (and refreezing) during cyro-sectioning this correlation should be lost as was found after intentional melting or frozen thin sections. These observations demonstrate that there is neither a through section melting nor a growth of ice crystals during cryo-ultramicrotomy.     

Rapid cryofixation of rabbit muscle fibres after a temperature jump

We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non‐helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X‐ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15–30 K from ~278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen‐cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze‐substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed ‘cold’ (~278 K) and temperature‐jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix‐based layer lines, similar to the differences seen by X‐ray diffraction. We conclude that we have preserved the ‘hot’ structure and that cryofixation is sufficiently fast to prevent the transition back to the ‘cold’ state.     

Ice crystal damage in frozen thin sections: freezing effects and their restoration

Thin sections of unfixed kidney, fast frozen without cryoprotectants, were fixed in osmium tetroxide vapour directly after freeze drying or after 30 min in a moist atmosphere. Dry sections fixed in vapour showed ice crystal damage characteristic for the freezing procedure. This was demonstrated with freeze fracture replicas from the same preparation. Ice crystal holes were obscured in serial sections which were freeze dried and allowed to rehydrate in a moist atmosphere. The same ultrastructural appearance was observed in frozen sections brought to room temperature immediately after cutting. Frozen thin sections from unfixed tissue, if freeze dried, are very sensitive to atmospheric conditions and need some form of stabilization (e.g. osmium vapour fixation, sealing with an evaporated carbon film) before electron microscope images can be interpreted as representative for the frozen state. Restoration of ice crystal damage can occur by melting frozen sections or by rehydration of freeze dried frozen sections. Restoration phenomena will impair studies aimed at the localization of diffusible substances by autoradiography or X-ray microanalysis.     

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.     

Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

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.     

A new method for scanning electron microscopic visualization of dermal elastic fibres.

Autoclaving sections of dermis for 8 h followed by fixation, dehydration and xylol-air drying yields a pure preparation of elastic fibres for scanning electron microscopy which retains the native architecture of this component. Elastic fibres were intertwined in a complex fashion with numerous branches. Fibres were predominantly cylindrical in the upper dermis and became larger and more elliptical in the deeper dermis. This method produces a means to study of organization of elastic fibres in a variety of disorders in which dermal changes are prominent.     

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.     

Preparing sections of skeletal muscle for transmission electron analytical microscopy (TEAM) of diffusible elements.

Comparative morphological examination and elemental analysis were carried out in structural compartments of sections of skeletal muscles. These had been prepared either by conventional plastic embedding technique or by various methods of cryo-ultramicrotomy. The analyses were performed in a Philips EM 301 with an Edax energy-dispersive X-ray spectrometer. Spectra obtained from sections of plastic-embedded muscle depended on the reagents used for fixation and staining and were absent if these were omitted. Brief fixation with glutaraldehyde resulted in gross ionic changes, and sectioning of frozen material with trough liquid led to extraction of elements. Sections cut from unfixed and frozen muscle without trough liquid showed numerous peaks. (Mg, P, S, Cl, K, Ca). In the superficial parts of the fibres of freeze-dried sections reproducible spectral differences were found between different structures. Thus, rapid freezing of unfixed tissue, dry cutting in the frozen state, and freeze-drying should be the procedure of choice if data on diffusible ions are desired.     

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.     

Direct evidence of radial and tangential morphology of high-modulus aromatic polyamide fibres

Previous work, at foreign laboratories, essentially based on electron microscopy of longitudinal sections, has suggested a radial morphology for the aromatic poly-amide high modulus fibres; the present paper gives direct evidence of such a morphology, thanks to a special preparation technique which allows a great improvement in the quality of the cross-sections of these fibres. This is demonstrated for both a commercial ‘Du Pont de Nemours’ yarn sample ‘Kevlar 29’, and an experimental high modulus aramid RPT (Rhǒne Poulenc Textile) yarn. In the first case, Ag₂S insertion technique was used and permitted one to see, on the cross-sections, an alternation of dark and clear radial bands, again with a tendency towards tangential splitting. In the second case the fibres were included into an amorphous polymer, a sample preparation technique which greatly enhances the quality of the cross-sections; optical microscopy showed the radial morphology fairly well; dark-field transmission electron microscopy—using the equatorial doublet of the electron diffraction pattern—allowed satisfactory resolution: both the radial, and occasionally the tangential, splitting, and the size of the cross-sectioned crystallites were easily revealed: these crystallites appear as isodiametric bright particles c. 15 nm in lateral size.     

Localization of acid and alkaline phosphatase

Ultrathin frozen sections of glutaraldehyde fixed yeast cells have been successfully used for the demonstration of acid and alkaline phosphatase. Acid phosphatase was localized over the cell wall of both the mother cell and the bud as well as over the newly forming cross wall (septum). Cytoplasmic vesicles (vacuoles, lysosomes?) located close to the cell wall showed a positive reaction for acid phosphatase as well. After 3 h glutaraldehyde fixation an activation of the nuclear acid phosphatase was observed. Lead precipitates were predominantly found over the nucleolar material of ‘resting’ and budding cells. Alkaline phosphatase could be demonstrated in the ‘yeast-mesosome’ and within the plasmalemma invaginations. After separation of the bud, small vesicles, probably derived from the endoplasmic reticulum showed a strong positive reaction for alkaline phosphatase. In frozen sections incubated for alkaline phosphatase, non-specific lead precipitates were found in the nucleus and along the plasmalemma invaginations.     

A procedure for identifying textile bast fibres using microscopy: Flax,nettle/ramie,hemp and jute

Identifying and distinguishing between natural textile fibres is an important task in both archaeology and criminology. Wool, silk and cotton fibres can readily be distinguished from the textile bast fibres flax, nettle/ramie, hemp and jute. Distinguishing between the bast fibres is, however, not easily done and methods based on surface characteristics, chemical composition and cross section size and shape are not conclusive. A conclusive method based on X-ray microdiffraction exists, but as the method requires the use of a synchrotron it is not readily available. In this paper we present a simple procedure for identifying the above mentioned textile bast fibres. The procedure is based on measuring the fibrillar orientation with polarised light microscopy and detecting the presence of calcium oxalate crystals (CaC₂O₄) in association with the fibres. To demonstrate the procedure, a series of fibre samples of flax, nettle, ramie, hemp and jute were investigated. The results are presented here. An advantage of the procedure is that only a small amount of fibre material is needed.     

Microstructural characterization in diffusion-bonded SiC/Ti–6Al–4V composites

The microstructural evolution during the diffusion bonding consolidation of a Ti–6Al–4V/SiC fibre composite was investigated by optical, scanning and transmission electron microscopy. The effects of processing parameters, particularly temperature, on the microstructures of the matrix and the fibre and their bonding were considered. Processing at too high a temperature can result in growth of SiC crystals in the fibre in addition to rapid interfacial reaction, while interfacial bonding cannot be established if the temperature is too low. Various defects can be caused by inadequate fabrication practices. These include micro-pores, matrix-cracking, cracking, bending and impingement of fibres, and heterogeneous fibre distribution. Methods for avoiding these are discussed. A defect-free and uniformly distributed fibre composite can only be achieved by optimizing the processing parameters (such as temperature, pressure, time and cooling rate) and adequately combining fibre spacing and matrix thickness with accurate fibre alignment.     

Surfaces of cryosections: is cryosectioning ‘cutting’ or ‘fracturing’?

In order to determine if cryosectioning involves ‘fracturing’ or ‘cutting’ we examined the surfaces obtained in cryosectioning by a metal-replicating procedure commonly used in freeze-fracture microscopy. Platinum-carbon replicas were made of the surfaces of both the sections and the complementary surfaces of the sample stubs from which the sections were cut. When samples of frozen red cells were sectioned at ?120°C with large knife advancements (1 μm), the chips produced did not resemble sections. Membrane fracture faces, produced by splitting of the lipid bilayer, were found in electron micrographs of replicas of the sample stubs. This demonstrates that a cryomicrotome can be used to produce large intact replicas. When dull knives were used with small knife advancements, both smooth and fractured regions were found. The sections produced with dull knives had a snowflake appearance in the light microscope. When sharp knives were used with small advancements (0·1 μm), replicas of the surfaces were free of fracture faces and the sections had a cellophane-like appearance in the light microscope. Therefore, in cryosectioning a different process other than ‘fracturing’ is responsible. This ‘cutting’ process may be micromelting of a superficial layer by the mechanism of melting-point depression from the pressure exerted by the sharp edge of the knife.     

An anti-roll plate for flattening of ‘dry’ ultrathin frozen sections

A simple anti-roll plate has been designed for use in preparing ‘dry’ ultrathin frozen sections. The plate, which is made of a strip of a glass cover slip and attached close to the edge of a glass knife, prevents curling of the sections.     

Immersion fixation of rapidly frozen,untreated tissues and suspensions of cells including spermatozoa

Small pieces of tissue, and cell suspensions in plastic artificial insemination (AI) ‘straws’, were frozen rapidly in Freon-12 at — 155°C, without pre-treatment. Peripheral fragments were thawed directly in 2% glutaraldehyde at 0°C, and processed for transmission electron microscopy (TEM) studies. Preservation of ultrastructure was satisfactory, and freezing artifacts were minimal.     

Microstructure-property relationships in silicate-matrix composites

Electron microscopy and associated analytical spectroscopic techniques have been used to characterize interfaces in SiC-fibre/silicate-matrix composites. Interface structure, formed via reaction during hot-press fabrication, is a function of time, temperature, matrix composition and fibre type. Interfaces with Nicalon or Tyranno fibres vary from amorphous carbon in fine precipitated form to continuous graphitic layers. Interface behaviour in a stressed composite, and hence the matrix microcracking stress, is a sensitive function of microstructure. Interface debond and shear properties have been assessed using indent-based ‘push-down’ and ‘push-through’ tests using a specially developed instrument within a scanning electron microscope. This uses piezoload measurement and translation, and is capable of dynamic image recording of the indentation sequence. Interface micromechanical (indent) measurements have been correlated with structure and macromechanical response in bend testing for a range of fibre/matrix types, processing and post-processing thermal treatments. An example is also given of interfaces prepared by fibre precoating.