Quantitation of shrinkage during preparation for scanning electron microscopy: Human lung

Human lung tissue is found to shrink considerably with preparation for SEM. Fifty-one blocks of glutar-aldehyde-fixed and inflated lung, approximately 2.5 cm × 2.5 cm × 1 cm, shrank a mean of 19% (± 4.0% SD) linear dimension through post fixation, dehydration and critical point drying. Shrinkage with fixation was not measured. Blocks of lung were observed to shrink equally in length (L) and width (W), L = 19.4% ± 2.7 SD, W = 19.0% ± 4.0 SD. Final shrinkage was the same whether samples were dehydrated in acetone or ethanol, although with acetone more of the shrinkage occurred during the dehydration process and less occurred during critical point drying.     

A quantitative study of the preparation of rabbit aortic endothelial cells for scanning electron microscopy

Quantitative studies were done with the scanning electron microscope (SEM) on aortic endothelial cells from ten rabbits. Of these, five were plastic casts and five were dehydrated with three different, but standard, techniques. The results indicated that all forms of dehydration caused significant shrinkage artefacts and that these were different in different directions in both the thoracic and abdominal aorta. The greatest shrinkage was found with the critical point drying technique, 45% in the abdominal aorta and 31% in the thoracic aorta. In the abdominal aorta this shrinkage was mainly due to a shrinkage in length (36%) rather than a shrinkage in width (15%). In comparison, in the thoracic aorta critical point drying resulted in a 15% shrinkage in length and a 19% shrinkage in width. Air drying and alcohol dehydration caused considerable shrinkage (29% and 18% respectively in the thoracic aorta, 29% and 36% respectively in the abdominal aorta). Directional differences were also found with these techniques, for instance alcohol dehydration in the thoracic aorta resulted in 0% shrinkage in length and 18% shrinkage in width.     

Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying.

The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.     

Air-drying of human leucocytes for scanning electron microscopy using the GTGO procedure

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO-treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO-treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO-AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

Identification and study of the same cell in monolayer culture by different methods of light microscopy,scanning, and transmission electron microscopy. Application for cryodamaged cells examination

Cells were cultivated on transparent conductive substrates, glass slides coated with indium oxide; individual cells were marked with a diamond indentor. Cell cultures were frozen (–15°C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast, fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM; after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.     

Low-temperature scanning electron microscopy of frozen hydrated mouse lung

A method is presented by which water is preserved as ice during examination of the lung in the scanning electron microscope (SEM). The lung need only be inflated, frozen, transferred to the microscope and examined with the electron beam. Chemical fixation, solvent dehydration, and drying are not necessary. The low-temperature SEM of Pawley and Norton [11] maintains lung at ?180° C, nearly liquid nitrogen temperature, for extended periods with a Joule-Thomson refrigerator built into the stage. It has an integral high-vacuum preparation chamber attached to the microscope column which allows serial fracture, low-magnification stereo light microscopy, radiant etching, and evaporative coating with gold or carbon. The stage can be tilted from 0° to 45° and rotated a full 360°. It is demonstrated that the air-liquid interface in the lung can be examined and that low-temperature SEM can be used to investigate the shape of alveoli, the patency of the pores of Kohn in the hydrated state, and the shrinkage and distortion of lung with drying.     

Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy

This paper describes the results of experiments in which the volume changes in mouse embryo limb samples were followed more or less continuously after fixation through dehydration and critical point drying, with in some instances data relating to post critical point drying shrinkage. 14 and 15 day p. c. mouse embryos were fixed in 3 % glutaraldehyde in cacodylate buffer and stored in this fixative until use. Single specimens were studied using a Quantimet image analysing computer to record the changes in projected area of the unmounted specimens as they were passed through the usual series of reagents according to various commonly used dehydration schedules. The area changes were converted to volume changes for the purposes of presentation in this paper. The Quantimet system could not be used to follow volume changes in the CPD bomb so that most experiments detail the volume in the intermediate fluid before CPD and the size of the specimen immediately after it was removed from the CPD bomb. A few experiments were conducted in which the specimens were measured whilst they were in the CPD bomb. The measurements relating to dehydration and CPD procedures were compared with measurements of air dried and freeze dried specimens. All three drying methods cause considerable shrinkage: freeze drying to 85 % of the glutaraldehyde fixed tissue volume; critical point drying to 41% (after 24 h); and air drying from a volatile solvent to about 18% of the fixed tissue volume. Air drying from water caused a shrinkage to about 12% of the original volume. There was no significant difference between the various commonly used CPD schedules or between GA only and GA + Os O₄ fixed tissue. CPD via cellosolve and CO₂ caused substantially more shrinkage than other methods. Dimensional changes during specimen preparation are probably associated with changes in shape and in relative relationships between organelles, cells and tissues having different compositions. This should be borne in mind by all those interpreting scanning electron micrographs of dried animal soft tissue specimens.     

A multisample chamber for dehydration and critical point drying

The principles and methods for constructing an improved chamber for dehydration and critical point drying of multiple biological samples are described. The specimen chamber design is based on vertical positioning of the electron microscope grids or coverslips and permits minimal perturbation of laminar solvent flow past the specimens. This condition is requisite for optimal exposure of samples to solvents, which is necessary for complete dehydration and drying. Fragile samples, including chromosomes, critical point dried in the multisample chamber demonstrate crisp, well-preserved, three-dimensional morphology.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

Application of polystyrene disk substrates in cellular cultivation methods: generalized specimen preparation protocol for scanning electron microscopy

Sample preparation for scanning electron microscopy (SEM) may vary by cellular type, composition and method of cultivation. It has been proposed here that a generalized method of sample preparation may be applied for the visualization of bacteria, fungi, and human cellular tissue without modification of protocol between cell types. The following protocol was developed to incorporate polystyrene disk substrates in the simplification of sample preparation for SEM and reduce the possibility of processing artefacts. The proposed method of preparation may be applied to samples grown in either liquid or solid cultural medium regardless of cell type. With the proposed protocol, centrifugation, isolation and critical point drying are not required, therefore increasing specimen integrity. The incorporation of polystyrene disks showed positive cellular adhesion and applications in SEM for bacterial, fungal and human neuronal tissue. In addition, the simplicity of the proposed protocol is highly adaptable and may be further incorporated to visually analyse the effects of antifungals, antibiotics and disease pathogenesis through pathogen–host interactions. The proposed method of specimen preparation was incorporated in either liquid or solid state growth mediums during the cultivation of the selected cellular samples and revealed great promise in the preservation and visualization under the scanning electron microscope.     

Iron-polyethylene glycol impregnation: An attempt to reduce shrinkage of specimens in SEM preparation

Glutaraldehyde-fixed HeLa cells were soaked in a mixture of fine cationic iron colloid and polyethylene glycol, immersed in tannic acid solution containing guanidine hydrochloride, and stained with osmic acid. The treated cells showed little shrinkage in the scanning electron microscope even after ethanol dehydration and CO₂ critical point drying. On the assumption that every HeLa cell maintained contact with each other, preservation rate was computed as 0.975 × 0.0033 in linear dimension. Microvilli on the cell surface were well preserved, and few undersirable deposits were noted on the specimen surface. This treatment was also applicable to bulk staining of tissue blocks, such as rat kidneys. The podocyte foot processes and endothelial micropores of the glomerulus were well preserved; the epithelial cells of the Bowman's urinary capsule were not collapsed; the microvilli of the brush border of the proximal convoluted urinary tubule kept their ordinary length (2 μm).     

A comparative survey of techniques for preparing plant surfaces for the scanning electron microscope

A comparative investigation of techniques for the preparation of soft botanical tissue for the scanning electron microscope has been carried out using the leaves and petals of Pelargonium zonale as test specimens. Twelve different preparative procedures involving combinations of fixation, dehydration, air drying, freeze drying, critical point drying, coating methods, replicas and a temperature controlled specimen stage were tested.     

Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver

The dimensional changes of small cubes of glutaraldehyde fixed mouse liver tissue were measured using a light microscope image projected into the Quantimet 720 Image Analysing computer system. The dimensional changes occurring in the critical drying bomb could be followed at all stages when violent turbulence was not occurring. The results show that liver tissue blocks shrink in four stages whilst in the critical point drying bomb: (1) during substitution of the intermediate solvent with the transitional fluid; (2) when the transitional fluid is warmed above the critical temperature; (3) when the transitional fluid, now a gas, is allowed to escape from the CPD bomb – the rate of shrinkage increasing as atmospheric pressure is approached; (4) at atmospheric pressure when all the gas has been allowed to escape from the bomb. Taken together with the authors' previous findings, it would seem that substantial shrinkage of animal soft tissue specimens must occur whilst they are undergoing “critical point drying”. This fact should be taken into account when interpreting SEM images of CPD tissues.     

A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

Preparation of cultured mammalian cells for transmission and scanning electron microscopy using aclar film

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.     

Description of a coverslip holder for,and observations on,the critical point drying of cell cultures

The elimination of artefact during the preparation of cell cultures for scanning electron microscopy is difficult. Collapse of cellular projections, cytoplasmic cracks, perforations and fracturing of cell-cell processes and cell-substrate attachments occur during fixation, dehydration and critical point drying. Coating and storage may cause further artefact. A specimen holder which serves to minimize turbulence in the critical point dryer and which allows for the simultaneous processing of up to five coverslips, as well as a reproducible technique for the preparation of cell cultures are described.     

Liquid substitution: A versatile procedure for SEM specimen preparation of biological materials without drying or coating

Certain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo-preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.     

Ultra‐thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin‐infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge‐like morphology of nondistinguishable intracellular compartments. Resin‐infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell–cell and cell–surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra‐thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three‐dimensional features by scanning electron microscopy.     

Improved ultrastructural preservation of yeast cells for scanning electron microscopy

The processing of yeast cells for scanning electron microscopy by conventional sequential fixation with glutaralde-hyde and osmium tetroxide and subsequent dehydration and critical point-drying caused pronounced deformation and visible shrinkage in all basidiomycetous and ascomy-cetous yeast strains studied. The mean cell diameter decreased to nearly 60 and 70%, respectively. After an additional sequential fixation with 1% tannic acid and 0–5% uranyl acetate the cell shrinkage was significantly reduced, but the most important result was a considerable reduction of wrinkling and deformation of the yeast cells.