Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO₄ fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO₄ fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO₄ fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.     

Form and distribution of actin and myosin in non-muscle cells: a study using cultured chick embryo fibroblasts.

Attempting to throw light on the mechanical basis of movement of non-muscle (cf. muscle) cells, the present work aims to determine the form and distribution of actin and myosin in chick embryo fibroblasts. These cells were cultured on formvar, fixed in glutaraldehyde then osmium tetroxide vapours, dehydrated, critical-point dried and examined, in toto, in the electron microscope (EM). Stereoscopic pairs of micrographs were studied to define more exactly the form and distribution of cytoplasmic filaments topographically associated with deformations of the cell surface and with organelle movements through the cytoplasm. Permeating the cytoplasm, interconnecting long and short filaments closely surrounded all organelles, linked with microtubules and polyribosomes and joined to the plasma membrane. These filaments, which varied greatly in width (2-13 nm) were closely associated with large numbers of 'comma-shaped' globoid bodies of approximately 15 nm diameter. Attempting to establish the identity, form and distribution of cytoplasmic myosin, cultured cells were extracted with a cold (4 degrees C) glycerol/pyrophosphate solution for 24 h before being fixed and critical-point dried. EM examination of these cells revealed a residual three-dimensional network of branching and anastomosing 4-13 nm diameter smooth filaments, devoid of fine (2 nm) filaments and globoid bodies. Examination of fixed, critical-point dried, skeletal muscle heavy meromyosin showed globoid structures similar in form and size to the globoid bodies found in cultures fibroblasts. Similarly fixed and critical-point dried paracrystals of actin, polymerized in the presence of Mg2+, appeared as branching interconnecting filaments which, in form and dimensions, resembled the network filaments observed in pyrophosphate-extracted cells. It is concluded that the pyrophosphate-extractable globoid bodies found in cultured fibroblasts represent monomers of myosin, that the broader filaments to which these attach represent actin in Mg2+ paracrystalline form and that the various subcellular movements are brought about by interactions between the two, analogous to those occurring in muscle cells.     

Surface topography and intracellular organization of human cells in suspension as revealed by scanning electron microscopy

This report presents a new procedure to study the ultrastructure of human cells in suspension by means of scanning electron microscopy. Living cells were maintained in suspension within cell culture flasks located on a rotating tilting table within an incubator. These cells were injected into warm glutaraldehyde/formaldehyde fixative. After washing in buffer, fixed cells were attached to propylamine-derived glass carriers. However, saturating free aldehyde groups of fixed cells and blocking amine groups of the derived glass carriers prevented cells from attachment to these carriers. Thus, we postulated that bonds between the fixed cell-free aldehyde groups and the carrier amine groups were responsible for cell-to-carrier attachment. Fixed cells attached to the carriers were subsequently dehydrated, dried, and coated for surface topography studies. For studies of internal cell organization, these attached cells were immersed in agar or gelatin as extracellular embedments and infused with sucrose or polyvinyl-pyrrolidone as cryoprotectants. Cells then were frozen and fractured. Fractured cells were either thawed, dehydrated, critical point dried, and ion beam sputter-coated, or freeze-substituted, dried, and planar magnetron sputter-coated. Finally, cell preparations were observed in the scanning electron microscope. Due to high cell attachment yield, both approaches samples observed in the electron microscope were representative of the entire cell population.     

Field-emission scanning electron microscopy of the internal cellular organization of fungi

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.     

Dilute aldehyde treatment on aldehyde-fixed cells for observing chromosomes in situ with the scanning electron microscope

A new preparation method is introduced to reveal intracellular structures in the scanning electron microscope and its application to mitotic cells in root meristems of Vicia faba is demonstrated. The root tips are fixed with a mixture of formaldehyde and glutaraldehyde and the fixed tissues are frozen and fractured in liquid nitrogen. They are then incubated successively in dilute solutions of aldehyde (formaldehyde or glutaraldehyde) and osmium tetroxide. By this treatment, the excess cell-matrix is removed from the fractured surface of the cell, and a deep view into the cell-interior can be obtained with the scanning electron microscope. Varied levels of substructure are observed on the surface of chromosomes.     

Cutting teeth in the SEM

An SEM was used to observe and record dental tissues as they were being cut. Sound human deciduous and permanent teeth were stored in 70% ethanol until required, then soaked in water, superficially dried and screwed to the SEM specimen stage through small drill holes made when they were wet. Many specimens were frozen and studied at cryogenic temperatures so that they would not become dehydrated. Edges used to cut the teeth included steel and tungsten carbide fashioned to resemble clinical cutting instruments, and diamond ultramicrotome knives and burs. The cutting tools were held either in a micromanipulator or a rigid tool post clamped to the specimen stage. The finest control was obtained by moving the specimen with the usual stage controls. SEM was conducted at 3 or 5 kV using TV speed scanning on the uncoated samples. All experiments were video-taped. 3-D control was difficult with a mono image and a real-time stereo system was therefore developed. Continuous, flowing sections of enamel could be obtained using diamond knives to cut the prism-free, surface zone tangentially. Thin sections of dentine, cement and bone curled up as they were cut, thus demonstrating permanent deformation. Subsurface enamel always fractured as it was cut, either locally as the tissue passed over the knife edge or tore out beneath the plane of the knife or by larger fragments cleaving off at larger distances ahead of the knife. Appearances were characteristic of prism orientation with respect to cutting direction. No anisotropy of cutting behaviour was found with dentine or bone: These tissues only fractured when thicker sections were taken. The SEM methods employed here can be usefully applied in the study of other materials.     

A method for TEM visualization of the extracellular matrix three-dimensional organization in tissues

A method for obtaining high-resolution three-dimensional images of the extracellular matrix organization in tissues is described. It consists of TEM observation of rotary-shadowed platinum–carbon replicas obtained from critical-point dried resinless sections of polyethylene glycol-embedded specimens. The procedure is simple and rapid, with high rates of sample recovery. An example of its application to EM immunocytochemistry (fibronectin localization) is presented. The utilization of the method to demonstrate cell-extracellular matrix relationships, and its limitations in the study of cells are discussed.     

Dimensional stability in tissues dehydrated with 2,2-dimethoxypropane for electron microscopy

Dimensions of tissues fixed in glutaraldehyde-osmium tetroxide mixture, osmium tetroxide and uranyl acetate and then dehydrated in 2,2-dimethoxypropane (DMP) were measured using transmission and scanning electron microscopy. Rat cardiac muscle, kidney and other tissues were examined in this study. The mean dimensions of characteristic ultrastructural features of material prepared by this method are similar or larger than those reported in the literature for conventionally processed samples. Critical point drying of specimens dehydrated with DMP does not produce abnormal shrinkage. Simultaneous primary fixation of lipids and proteins in a glutaraldehyde osmium tetroxide mixture and omission of the water wash after uranyl acetate appear to be important in stabilizing the tissue for rapid dehydration. The rapid reaction of DMP and water yielding the products acetone and methanol does not appear to denature tissue components to a greater extent than conventional solvent exchange dehydration.     

Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls

This article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three-dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and anti-actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.     

Preparation of cultured cells for SEM: air drying from organic solvents.

Air evaporation from organic solvents of differing polarities and surface free energies was used in the preparation of cultured murine peritoneal macrophages for scanning electron microscopy (SEM). The surface structural features of these cells were compared to the surfaces of similar cells prepared by the critical-point procedure. In general, all organic solvents produced a marked collapse of cell structure resulting in an increase in surface irregularity. Fracture surfaces and sharply defined contours including numerous flaps and ridges were characteristic of the non-polar solvent dehydrated samples. Polar solvent dehydrated samples, including those dried from solvents of low surface free energy, exhibited a secondary flowing and settling of the cell membrane. Primary collapse was apparent but cell contours were smoothed and rounded. Overall cell shape and cell-to-cell relationships were retained regardless of solvent type. It is suggested that solvent evaporation may prove useful in certain cases, though investigators are advised to use caution when interpreting the results obtained by such procedures.     

X-ray microanalysis of ultrathin frozen and freeze-dried sections of human sperm cells

X-ray microanalysis of air dried and ultrathin frozen and frozen dried sections of human sperm cells has indicated a large deviation of elemental composition between cells in a single sample and between samples. Analysis of air dried cells indicates a similar subcellular elemental distribution to that found in sectioned material. Sodium to potassium ratios are found to be similar in both preparations. Air drying is considered a valid method for the preparation of sperm cells for analysis.     

Quantification of plant cell coupling with three-dimensional photoactivation microscopy

Plant cells are directly connected by plasmodesmata that form channels through the cell wall and enable the intercellular movement of cytosolic solutes, membrane lipids and signalling molecules. Transport through plasmodesmata is regulated not only by a fixed size-exclusion limit, but also by physiological and pathological adaptation. To understand plant cell communication, carbon allocation and pathogen attack, the capacities for a specific molecule to pass a specific cell-wall interface is an essential parameter. So far, the degree of cell coupling was derived from frequency and diameter of plasmodesmata in relevant tissues as assessed by electron microscopy of fixed material. However, plasmodesmata functionality and capacity can only be determined in live material, not from electron microscopy, which is static and prone to fixation artefacts. Plasmodesmata functionality was a few times assessed using fluorescent tracers with diffusion properties similar to cytosolic solutes. Here, we used three-dimensional photoactivation microscopy to quantify plasmodesmata-mediated cell-wall permeability between living Cucurbita maxima leaf mesophyll cells with caged fluorescein as tracer. For the first time, all necessary functional and anatomical data were gathered for each individual cell from three-dimensional time series. This approach utilized a confocal microscope equipped with resonant scanner, which provides the high acquisition speed necessary to record optical sections of whole cells and offers time resolution high enough to follow the kinetics of photoactivation. The results were compared to two-dimensional measurements, which are shown to give a good estimate of cell coupling adequate for homogenous tissues. The two-dimensional approach is limited whenever tissues interfaces are studied that couple different cell types with diverse cell geometries.     

Advantages and pitfalls of using free‐hand sections of frozen needles for three‐dimensional analysis of mesophyll by stereology and confocal microscopy

The anatomical structure of mesophyll tissue in the leaf is tightly connected with many physiological processes in plants. One of the most important mesophyll parameters related to photosynthesis is the internal leaf surface area, i.e. the surface area of mesophyll cell walls exposed to intercellular spaces. An efficient design‐based stereological method can be applied for estimation of this parameter, using software‐randomized virtual fakir test probes in stacks of optical sections acquired by a confocal microscope within thick physical free‐hand sections (i.e. acquired using a hand microtome), as we have shown in the case of fresh Norway spruce needles recently. However, for wider practical use in plant ecophysiology, a suitable form of sample storage and other possible technical constraints of this methodology need to be checked. We tested the effect of freezing conifer needles on their anatomical structure as well as the effect of possible deformations due to the cutting of unembedded material by a hand microtome, which can result in distortions of cutting surfaces. In the present study we found a higher proportion of intercellular spaces in mesophyll in regions near to the surface of a physical section, which means that the measurements should be restricted only to the middle region of the optical section series. On the other hand, the proportion of intercellular spaces in mesophyll as well as the internal needle surface density in mesophyll did not show significant difference between fresh and frozen needles; therefore, we conclude that freezing represents a suitable form of storage of sampled material for proposed stereological evaluation.     

High-resolution scanning electron microscopy of the surface of red blood cells

Examination of normal human red blood cells with a high (2.5 nm) resolution scanning electron microscope revealed a definite surface pattern. The pattern was slightly more pronounced in specimens dried in air after fixation than in those dried by the critical-point method. There was a radial arrangement of filament like elevations at the periphery of the red cell and a more reticular pattern to wards the centre of the cell. The appearances probably represented a network of elenin fibres lying just under the surface of the red cell.     

Routine cryofixation of plant tissue by propane jet freezing for freeze substitution

Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.     

Preparing and analyzing fractured archaeological fibers

A technique was developed to prepare archaeological fiber cross sections for electron microscopic examination and x-ray analysis. Use of this new method allows chemical and morphological information to be obtained from the interior of a single fiber or yarn. Fibers are fractured while frozen and then freeze dried. Following mounting and carbon coating, fibers are examined by scanning and backscatter electron microscopy and then analyzed by using energydispersive spectrometry. Elemental distribution is mapped by using image-processing software. In this report, the described technique is employed in the examination of ancient fibers from three different long-term storage environments (moist buried, dry buried, museum stored). Data obtained by examining the interior of fibers such as these provide insight into the conditions of a fiber's growth, the treatments applied during the fiber's processing and use, and the conditions in which the fiber was stored.     

Techniques for obtaining and observing complementary images with a low-temperature field emission SEM and subsequent comparison of the identical cells in freeze-etch replicas viewed with a TEM

Initial evidence shows that low-temperature (LT) field emission scanning electron microscopy (FESEM) provides high-resolution complementary images of frozen, fractured biological tissues and that the coating or replicas from the tissues can be recovered and viewed in the TEM to compare the identical cellular structures. To observe frozen specimens, a Hitachi S-4000 FESEM was equipped with an Oxford CT 1500 Cryotrans System. The standard Oxford specimen carrier was modified to accommodate a Denton complementary freeze-etch cap that would hold up to 6 hinged 24K gold specimen holders. This combination allowed observation of complementary images of sputter-coated, freeze-etched biological specimens at magnifications up to X30,000. Resolution of sputter-coated images was also compared with that from evaporative coatings. Use of high-vacuum evaporation of Pt/C in conjunction with LT-FESEM provided useful resolutio up to X100,000. In addition, after these specimens were observed in the LT- FESEM, their coating, which consisted of a freeze-etch replica, could be recovered from the frozen tissues and subsequently observed in the TEM at even higher resolutions. Consequently, complementary images of frozen, fractured, fully hydrated cells could be observed in the LT-FESEM and then compared to the complementary images of the identical cells that were present in the replicas, which were recovered from the frozen specimens, and subsequently observed in the TEM. Being able to evaluate, compare, and contrast data from these two different EM imaging techniques as well as from complementary surfaces, not only provides additional information about the ultrastructure of a specimen, but also helps to assess the resolution of coating films, the presence of contaminants and the three-dimensional distortion in replicas.     

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.     

Cryo-sectioning and chemical-fixing ultramicrotomy techniques for imaging rubber latex particle morphology

Two methods adapted from biological microscopy are described for a new application in imaging the morphology of rubbery latex particles. In the first method, a drop of latex is frozen in liquid nitrogen, sectioned with a diamond knife and vapour-stained with osmium tetroxide, then viewed by transmission electron microscopy. When applied to latexes made by emulsion polymerization of methyl methacrylate in a natural rubber latex seed, inclusions are clearly visible. A chemical fixation method is then described for imaging the morphology of such rubbery latex particles. Glutaraldehyde is added to the latex, followed by osmium tetroxide. The sample is then dehydrated in ethanol, epoxy resin added, and the sample cured, ultramicrotomed, and imaged with transmission electron microscopy. An inclusion morphology is again clearly seen.     

Examination of cell suspensions prepared by cryobiological techniques*

Ultrathin frozen sections have been obtained from single cells both in the fixed and native state. For fixed material a rapid method is described which yields excellent structural preservation. The preparation is based on brief treatment with glutaraldehyde, polyvinylpyrrolidone and ‘encapsulation’ in gelatin. Ultra-thin frozen sections prepared after this technique seem to be especially suitable for cytochemical work. Unfixed testis has been also cut with a dry knife. The contrast in this completely untreated section is sufficient to discern the different parts of spermatozoa.