Freeze-dried and resin-embedded biological material is well suited for ultrastructure research

Transmission electron micrographs of different biological material, cryofixed, freeze‐dried and embedded in Spurr's resin, in Epon, or in Lowicryl, are presented. The structure preservation obtained either without or with application of chemical fixatives after drying showed that freeze‐dried embedded specimens are particularly well suited for new morphological, immunocytochemical and microanalytical studies aimed at detecting the life‐like subcellular distribution of mobile macromolecules and ions. The results also indicate that the removal of cell water by freeze‐drying from the areas of best cryofixation is relatively slow. Ultrathin sections of well cryofixed biological material embedded after freeze‐drying in Spurr's resin or Epon reveal cellular plasma phases with very fine granularities and well defined membranes in negative contrast. This may be due to the preservation of the original structure of cellular macromolecules with a considerable amount of their hydration water. Sublimation studies with differently hydrated and cryofixed macromolecules are suggested to settle this issue.     

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.     

Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure

Caenorhabditis elegans is one of the most important genetic systems used in current biological research. Increasingly, these genetics‐based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function. Here, we present and review state‐of‐the‐art methods for both ultrastructural analysis and immunogold localization in C. elegans. For the initial cryofixation, high‐pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing. The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E. coli and/or yeast paste prior to freezing. The latter method facilitates embedding of C. elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography.     

Preparation of resin embedded unicellular organisms without the use of fixatives and dehydration media.

A method is presented for processing single cells for conventional ultrathin sectioning without the use of fixatives and dehydration media. The cells were fixed by a physical method--spray freezing--which provides extremely high cooling rates, needs no pretreatment with cryoprotective agents and is therefore assumed to maintain the in vivo morphology of the cell. Hitherto cells prepared in this way have been investigated exclusively by freeze etching. To combine the advantages of this method with those of conventional ultrathin sectioning we have processed spray frozen cells with widely varying water contents (spermatozoa and lymphocytes) by freeze drying at 188 K and vacuum embedding. When compared to conventional chemical fixation the differences found in ultrastructural preservation of spermatozoa using this kind of preparation were confined to the arrangement of spermhead membranes and middlepiece structures. Lymphocyte structure was much closer to that known from chemical preparation, the only differences being a denser cytoplasm, denser mitochondrial matrices and thicker plasma membranes. These differences are probably due to the absence of eluating and dissolving effects present in conventional chemical preparations. The ultrastructural preservation of spray frozen cells is not different after freeze etching or after freeze-drying and vacuum embedding. This indicates clearly that drying and resin embedding does not produce artefacts and that structural preservation is therefore limited by the quality of cryofixation. Therefore this method is considered a contribution to the problem of preservation of the in vivo assembly of cellular substructure. Furthermore it seems to be a potential basis for preparation of soluble or diffusible substances or cellular compounds which would be influenced by fixatives and dehydrating agents.     

Fast freezing device

A fast freezing device for cryofixation of biological material, without cryoprotection, has been developed based on metal freezing (Van Harreveld et al., 1964, 1974) combined with preceding sectioning (Bernard & Krigman, 1974; Van Harreveld & Fifkova, 1975) as well as freeze sectioning and freeze-etch replication (Dempsey & Bullivant, 1976).     

Of plants and other pets: practical aspects of freeze-substitution and resin embedding

Representative tissues from higher plants (e.g. developing pollen, somatic anther tissues from the monocotyledonous angiosperm Ledebouria) and mammalian cell cultures were successfully cryoimmobilized by means of high‐pressure freezing. Various substitution and embedding protocols were then evaluated considering the preservation of ultrastructural details, membrane staining, immunolabelling properties, as well as reproducibility and ease of use. Two types of recipe proved to be highly suitable for most applications, regardless of type, developmental stage or physiological conditions of the cells: (i) the best choice for morphology is still osmium in acetone (optionally supplemented with uranyl acetate) followed by embedding in Epon and/or Araldite; (ii) feasible approaches for immunocytochemistry are freeze‐substitution with ethanol containing uranyl acetate and formaldehyde, or with pure acetone (in the case of fixation‐sensitive antigens), followed by embedding with LR‐white acrylic resin; though being far from optimal, these combinations represent, in my opinion, an acceptable compromise between labelling intensity, section stability, structural preservation and health hazards. Notably, the patterns observed in Ledebouria were consistent with data obtained from a broad range of other specimens from all kingdoms (e.g. leaves and callus cultures from angiosperms, gymnosperm roots with their ectomycorrhizal fungi, mammalian cell cultures and eubacteria). Finally, a warning is given as to the extractive potentials of embedding resins (Spurr's mixture, LR‐white, but also Epon) being sometimes the cause of unacceptable artefacts, both in plant and in mammalian cells prepared by cryoimmobilization and freeze‐substitution.     

Candida albicans biofilms: comparative analysis of room‐temperature and cryofixation for scanning electron microscopy

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde‐based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room‐temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room‐temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze‐substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.     

An inexpensive device for freeze drying and plastic embedding tissues at low temperatures

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl® and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.     

Preparation of the nematode-trapping fungus,Arthrobotrys oligospora,for scanning electron microscopy by freeze substitution

A freeze-substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at — 20°C and critical-point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three-dimensional arrangement.     

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.     

Why low temperature embedding for X-ray microanalytical investigations?

Freeze-drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low-temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to ?50°C. Sections of low temperature embedded material can be cut dry at ?60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature. The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi-thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze-dried or freeze-substituted prior to embedding. Freeze-drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze-dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze-dried and embedded tissue.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. II. Other preparative methods

The loss of ¹⁴C-ethanolamine- and ³H-choline-labelled phospholipids from rat liver during preparation for electron microscopy by some less frequently used processing methods has been examined. Permanganate and formaldehyde-potassium dichromate fixation followed by Araldite embedding were investigated and five procedures involving embedding in water-miscible methacrylates (GMA). These procedures included a conventional method of dehydration and embedding in GMA, a low-temperature GMA embedding method, dehydration with ethylene glycol, freeze drying and freeze substitution. These results are compared with those obtained after conventional tissue preparation (presented previously, Cope & Williams, 1969). Formaldehyde-potassium dichromate compared favourably with the conventional procedures for the preservation of both phosphatides, especially phosphatidyl ethanolamine. Permanganate fixation was much less effective. Severe loss of both phosphatides occured after freeze drying and freeze substitution in glutaraldehyde-alcohol. GMA is shown to be a more potent phospholipid solvent than ethanol under the conditions employed. Low-temperature embedding reduced the loss of phosphatidyl choline during embedding. Results obtained by scintillation counting were confirmed by grain counts on thick-section autoradiographs. No direct relationship between extraction and the electron-microscopic appearance of membranes was discernible. It is believed that membrane prominence is largely dependent upon the electron density of the surrounding cytoplasm rather than on the degree of phospholipid extraction.     

The potential of cryofixation and freeze substitution: observations and theoretical considerations

The theoretical and experimental evidence in favour of cryofixation and freeze-substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.     

Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples

Specimen preparation methods based on high‐pressure freezing and freeze‐substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy. However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast. By contrast, some efforts to increase membrane staining may lead to excessively granular staining. No one freeze‐substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis. However, one or more of the following protocols, perhaps with minor modifica‐tions, should yield satisfactory results in most cases. Freeze‐substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high‐resolution structural studies. Membranes are typically lightly stained but very well defined. Initial freeze‐substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes. Finally, initial trials indicate that freeze‐substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes. The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze‐substitution reagent.     

Cryofixation and ultra-low-temperature freeze-drying as a preparative technique for TEM

We have developed cryofixation and ultra-low-temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples. Two sample types were investigated. In the first, pure deuterium oxide (D₂O), or heavy water, was vapour condensed under vacuum conditions onto a gold-coated copper sample holder held at ?175 or ?110°C. Additionally, D₂O was slow-rate cooled from room temperature under an ultra-pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D₂O loaded water and ultra-rapid cooling by metal-mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen-hydrated cryosections, were amorphous, cubic, and hexagonal. Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor-controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle. D₂O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D₂O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D₂O samples. Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen-hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal growth during the drying procedure.     

Silver enhancement of Nanogold particles during freeze substitution for electron microscopy

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well‐fixed, well‐resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno‐localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron‐dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor‐mediated membrane traffic.     

Improved method of producing satisfactory sections of whole eyeball by routine histology

To overcome the loss of structural integrity when eyeball sections are prepared by wax embedding, we experimentally modified the routine histological procedure and report satisfactorily well‐preserved antero‐posterior sections of whole eyeballs for teaching/learning purposes. Presently histological sections of whole eyeballs are not readily available because substantial structural distortions attributable to variable consistency of tissue components (and their undesired differential shrinkage) result from routine processing. Notably, at the dehydration stage of processing, the soft, gel‐like vitreous humor considerably shrinks relative to the tough fibrous sclera causing collapse of the ocular globe. Additionally, the combined effects of fixation, dehydration, and embedding at 60°C renders the eye lens too hard for microtome slicing at thicknesses suitable for light microscopy. We satisfactorily preserved intact antero‐posterior sections of eyeballs via routine paraffin wax processing procedure entailing two main modifications; (i) careful needle aspiration of vitreous humor and replacement with molten wax prior to wax infiltration; (ii) softening of lens in trimmed wax block by placing a drop of concentrated liquid phenol on it for 3 h during microtomy. These variations of the routine histological method produced intact whole eyeball sections with retinal detachment as the only structural distortion. Intact sections of the eyeball obtained compares well with the laborious, expensive, and 8‐week long celloidin method. Our method has wider potential usability than costly freeze drying method which requires special skills and equipment (cryotome) and does not produce whole eyeball sections. Microsc. Res. Tech. 77:138–142, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Freeze-fracture electron microscopic and low temperature X-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL₂) structure. Only when the LDL₂ solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL₂ X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL₂ X-ray scattering curves, in part due to the effect of quenching upon the solvent. Freezeetching electron microscopy of the slowly cryofixed LDL₂ showed marked aggregation of the particles and an unusual morphological appearance. In contrast, after rapid cryofixation or cryofixation in the presence of glycerol, freeze-etch electron microscopy revealed well-isolated particles which had a knobby morphology. The results demonstrate that under certain conditions (in the presence of 75% glycerol) cryofixation results in minimal, if any, structural alteration of, at least, the LDL₂ lipid moiety. Further, this study underlines the more general conclusion that any high resolution structural study employing a cryofixation step must be interpreted with caution and the effect of cryofixation upon the sample structure need be evaluated by independent means.     

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.