Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water

Biological membranes are often poorly visible with the electron microscope after high‐pressure freezing and freeze‐substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high‐pressure frozen yeast cells, rat‐pancreas tissue and arthropod tissue were freeze‐substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1–5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze‐substitution with water‐free medium but good after addition of 5% water to the substitution medium.     

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers

High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

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.     

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.     

High-pressure freezing in the study of animal pathogens

High‐pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot‐and‐mouth disease virus and African swine fever virus by the use of high‐pressure freezing of infected cells. Foot‐and‐mouth disease virus particles are not detected in sections of conventionally immersion‐fixed infected cells, but when the cells are prepared by high‐pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high‐pressure freezing of virally infected cells: first, two sapphire discs frozen ‘face to face’ with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high‐pressure freezing followed by freeze‐substitution in acetone with uranyl acetate allows high‐sensitivity immunolabelling for these proteins.     

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.     

Pure optical contrast in scattering-type scanning near‐field microscopy

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO₄–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO₄–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO₄–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO₄ oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.     

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

We describe a method for high‐pressure freezing and rapid freeze‐substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed ‘face to face’ without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice‐crystal artefact. Freeze‐substitution at −90°C for 60 min in acetone containing 2% uranyl acetate, followed by warming to −50°C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.     

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.     

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.     

Electron microscopy of the early Caenorhabditis elegans embryo

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser‐induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time‐resolved fixation to arrest development at specific stages. The second method uses high‐pressure freezing of whole worms followed by freeze‐substitution (HPF‐FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high‐resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF‐FS and electron tomography. This procedure combines the advantages of time‐resolved fixation and superior ultrastructural preservation by high‐pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.     

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.     

The use of cyanobacteria as filler in nitrocellulose capillaries improves ultrastructural preservation of immature barley pollen upon high pressure freezing

The high pressure freezing (HPF) followed by freeze substitution technique has advantages over chemical fixation in the context of preserving sample ultrastructure. However, when HPF is applied to cultured pollen grains, the large intercellular spaces present lead to a poor level of ultrastructure preservation. We report here that the mixing of cyanobacteria with immature barley pollen grains succeeded in greatly reducing the volume of liquid present between the large pollen grains, and so improved the loading of the sample into a nitrocellulose capillary. The use of yeast or cyanobacteria paste to surround the filled capillaries was beneficial in speeding the transfer of heat during the freezing process. This modification of the HPF method resulted in a greatly improved level of ultrastructure preservation.     

An application of rapid freezing,freeze substitution–fixation for scanning electron microscopy

A combined technique of the rapid freezing, freeze substitution–fixation method and the osmium–DMSO-osmium method was devised. By this combined method we clearly observed the architecture of intracellular components in three dimensions. Morphological characteristics were generally similar to those of tissue prepared by the osmium–DMSO-osmium method but different in some respects. Mucigen droplets in intestinal goblet cells, for example, appeared as separated spheres, while in specimens prepared by chemical fixation they were observed as a mass of fused droplets. In the Golgi complex, all cisternae were extremely flat, although they usually dilated on the cis side after chemical fixation. Particles on the mitochondrial tubules of liver cells were well distinguished. They were mushroom shaped, as are those observed by negative staining. The combined method, that is, the rapid freezing, osmium–DMSO-osmium method, is thought to be effective for studying the true structure of intracellular components by scanning electron microscopy.     

Electron microscopic observation of photoreceptor cells in directly inserted anesthetized Drosophila into a high‐pressure freezing unit

The high‐pressure freezing (HPF) technique is known to cryofix water‐containing materials with little ice‐crystal formation in deep depths compared with other freezing techniques. In this study, HPF for anesthetized living Drosophila was performed by placing them directly on the carrier of the HPF unit and exposing them to light. Frozen Drosophila were freeze substituted, and their compound eyes were examined by transmission electron microscopy. The ultrastructures of ommatidia composed of photoreceptor cells were well preserved. The location of the cytoplasmic organelles inside the photoreceptor cells was observed. In some photoreceptor cells in ommatidia of the light‐exposed Drosphila, the cytoplasmic small granules were localized nearer the base of rhabdomeres, compared with those of the nonlight‐exposed Drosophila. Thus, HPF with the direct insertion of living Drosophila under light exposure into the HPF machine enabled us to examine changes to functional structures of photoreceptor cells that occur within seconds.     

High concentration of phosphorus is a distinctive feature of myelin. An X‐Ray elemental microanalysis study using freeze‐fracture scanning electron microscopy of rat sciatic nerve

We have used rat sciatic nerves submitted to freezing and freeze‐fracture to determine the elemental composition of small domains of the peripheral nerve studied at high resolution by scanning electron microscopy. We found that myelin of Schwann cells is unique in its high content in phosphorus (P) that was more than 10 times higher than P measured in any other cells. This high concentration in P makes myelin chemistry suitable of monitoring at the subcellular level using the herein described methodology. Microsc. Res. Tech. 78:537–539, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.