Preservation of EDTA-expanded grid-mounted chromosomes and nuclei for electron microscopy using a specially designed freeze-dryer

We describe methods for freezing and drying EDTA-expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole-mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the sepcimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N₂ (-210°C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical-point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.     

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.     

Digital photogrammetric quantification of surface area and volume on scanning electron micrographs of frozen hydrated lung tissue

A digital video plotter (DVP, Leica), the personal computer equivalent of an analytical plotter, was used to measure the coordinates of points chosen from stereo pair images of the surface of frozen hydrated lung imaged at magnifications of 2000 and 5000x with a low-temperature scanning electron microscope (SEM). Rat lung tissue was frozen in vivo with a liquid nitrogen cryoprobe under carefully controlled physiologic conditions. At slow freezing rates, water in the aqueous layer at the surface of the lung segregates into ice crystals (dendrites) which branch in the plane of the surface. Coordinates of points on dendrite surfaces were measured by the DVP and passed to TERRAMODEL (Plus III Software, a land modeling program) where they were used to generate a three-dimensional model, from which surface area and planimetric area of the lung surface were calculated. Additional measurements were made at the top and bottom of the ice structures and a Basic language program was written to calculate the volume of ice on the lung surface. Digital photogrammetry coupled with low-temperature SEM of frozen samples allows measurement of water and water-containing microstructures ubiquitous in biology.     

Cryofixation of plant tissues without pretreatment

Root tips from Sorghum and Dahlia were frozen without cryoprotection by dipping into nitrogen slush, rapid immersion in liquid propane and by the high-pressure method. Structural preservation of the samples was studied using freeze-fracture (FF) and freeze-substitution (FS) techniques for electron microscopy. It was found that most of the organelles were disrupted by freezing in nitrogen slush and that only the boundary beween the cytoplasm and the vacuole remained visible. If the samples were frozen by rapid immersion in liquid propane, small membraneous organelles, such as dictyosomes, were preserved in peripheral regions of the rhizodermal cells up to 10 μm below the surface of the tissue. Specimens frozen by the high-pressure freezing technique showed good ultrastructural preservation throughout the tissues up to a depth of more than 100 μm.     

Visualization of biological specimens by cryoHVEM

This article describes the operation and the characteristics of cryoHVEM imaging of biological specimens using a top-entry cryostage. The procedure for inserting frozen specimens into the microscope column is also presented. Whole mounts were thus observed under optimal imaging conditions by combining: (i) fixation by fast freezing for structure preservation without exposure to chemicals, (ii) observation in the hydrated (frozen) state or in the dried state without exposure to the atmosphere after the initial fixation by freezing, and (iii) ultrastructural visualization with the key imaging factors of resolution, penetration and beam-induced damage at their best by high-voltage electron microscopy.     

High-pressure freezing of epithelial cells on sapphire coverslips

Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage‐free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing. In a novel approach, we tested the suitability of sapphire discs as cell substrata for high‐pressure freezing. Frozen samples on sapphire were subjected to freeze‐substitution while in the same flat sample holders as used for high‐pressure freezing. We obtained cells that displayed an excellent preservation of fine structure. Because sapphire is a tissue culture substratum suitable for light microscopy, its use in combination with high‐pressure freezing could become a powerful tool in correlative studies of cell dynamics at light and electron microscopic levels.     

Solutions to difficulties encountered in low-temperature SEM of some frozen hydrated specimens: Examination of Penicillium nalgiovense cultures

Penicillium nalgiovense cultures, which are used in the food industry, were found to be collapsed when prepared by standard procedures for scanning electron microscopy. Neither freeze-drying nor critical point-drying preserved the structure of cultures grown on agar media. Cryofixation and preparation of frozen hydrated samples using the Hexland Cryotrans CT 1000 attachment in conjunction with an AMR 1000A scanning electron microscope yielded micrographs of uncollapsed structures which could be used for morphological characterization. Several additional steps had to be used in sample preparation to achieve satisfactory results. Samples were held in a humid chamber prior to freezing; growth substrate was trimmed as thinly as possible (less than 1 mm above the support); the sides of samples were painted with a conductive cement to their upper edge; and frozen samples were coated intermittently with gold sputtered in several 2-min bursts.     

Rapid freezing and electron microscopy for the arrest of physiological processes

An apparatus for the rapid freezing of tissue is described, which can be used for the electron microscopy of arrested physiological processes. The material is frozen by bringing it in contact with a silver surface cooled to liquid nitrogen temperature at reduced pressure. The freezing surface is protected from condensation of moisture and gases from the air by a flow of helium gas. The cooling of the specimen during its descent through the cold helium is not large enough to interfere with physiological processes. Freezing occurs very rapidly in the surface but is retarded to about 8 msec at a depth of 10 μm. The apparatus was used to freeze frog muscle during contraction.     

Transfer,observation and analysis of frozen hydrated specimens

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit.     

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.     

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.     

Morphological changes in mouse embryos cryopreserved by different techniques

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing.     

Electron microscopy of frozen water and aqueous solutions

Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray-freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X-ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm^?2. We propose a possible model for the mechanism of beam damage in aqueous solutions. The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.     

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.     

Cryofixation of epithelial cells grown on sapphire coverslips by impact freezing

Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire‐attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal‐mirror impact freezing. The potential of the Leica EM‐CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze‐substitution (SD‐FS unit) has been exploited. Subsequently, the SD‐FS unit has been tested for its use in methanol freeze‐substitution and low temperature embedding for immunoelectron microscopy. The structural preservation of Lowicryl HM20‐embedded cells has been assessed as being free of damage by large ice crystals.     

Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

非闭合电极电容层析成像传感器在冻土测试中的应用

为了实现电容层析成像技术对冻土冻结冰峰面的在线、非侵入测试,研制出了满足冻土测试要求的非闭合电极电容层析成像传感器,并对该种传感器的电容分布特性进行了实测;确定出了适合冻土测试的的高、低介电常数的标定物质;搭建了冻土一维冻结实验系统.对含水量8%的湿土样冻结过程的冰峰面迁移进行了电容层析成像测试,并利用IMNSNOF图像重建算法重建出了冻结过程各时刻的冻结截面物质分布图像,由图像可确定出冻土中已冻土、未冻区以及冰峰面的位置.电容层析成像测试结果与温度测试结果相吻合.     

Cryofracture of paraffin-embedded heart muscle cells.

A comparative study of internal cellular structures of the sheep ventricular myocardium has been conducted by scanning electron microscopy (SEM) and by transmission electron microscopy (TEM). Access to the cell interior for three-dimensional studies was obtained by cryofracturing paraffin-embedded tissue frozen in liquid nitrogen. For accurate localization of structures of special interest thick paraffin sections were examined in the light microscope (LM). Based on the information gained, it was possible to fracture the block in a desired plane. The fracturing was carried out by a light blow to a precooled scalpel held against the surface of the block, which was immersed in liquid nitrogen. After thawing and deparaffinizing at room temperature in several baths of xylene, the tissue pieces were critical point dried using CO2. As xylene was found to be miscible with CO2, it also served as an intermediate fluid. This method resulted in good preservation fo the myofibrils, mitochondria, sarcoplasmic reticulum and transverse tubules (T-tubules), which was confirmed by TEM studies of conventionally prepared tissue and of tissue originally prepared for SEM.     

The use of filter membranes for high-pressure freezing of cell monolayers

Rapid freezing of cells and tissues, followed by freeze‐substitution fixation and plastic embedding, has become a highly reliable method for preparing samples for imaging in the electron microscope. High‐pressure freezing is an efficient means of immobilizing suspensions of yeasts, thick pellets of mammalian cells, or small (< 0.5 mm) pieces of plant or animal tissue. Monolayers of cultured mammalian cells that are too thick for efficient immobilization by other modes of rapid freezing have also been successfully preserved by this method. Monolayer cultures are often important because they can be imaged by light microscopy (LM) both before and after their preparation for electron microscopy (EM). Additionally, some monolayer cultures serve as model systems for physiological processes, so it is important that cells under study can grow on a substrate that is both physiologically appropriate and convenient for EM processing. Here we describe a reliable method for preparing mammalian cell monolayers (PtK₁ and polarized MDCK) for EM. Our protocol results in good preservation of cellular ultrastructure, it is a useful companion to studies of cell physioloy and, with some limitation, is suitable for correlative LM and EM.