Polystyrene Embedding: A New Method for Light and Electron Microscopy

Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and give acceptable results.     

ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

The diamond knife "semi": a substitute for glass or conventional diamond knives in the ultramicrotomy of thin and semi-thin sections

The diamond knife "semi" for ultramicrotomes was originally designed by its manufacturer (DIATOME S.A.), for cutting semi-thin sections from 0.2 micron to 2.0 micron. Cutting tests of Epon-embedded material (nervous system, myelin sheat) with this knife have shown that the quality of semi-thin sections is equivalent or better than that obtained with a glass knife, and much time could be saved during the microtomy of serial sections. The quality of thin sections (0.07 micron to 0.12 micron) is excellent and comparable to that obtained with a conventional diamond knife. Furthermore, when adjacent sections are cut thin and semi-thin (for immunochemistry or high voltage microscopy), both are excellent in that they are of uniform thickness. In conclusion, this tool has advantages for both light microscopy and transmission electron microscopy.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

ULTRATHIN FROZEN SECTIONS : II. Demonstration of Enzymic Activity

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35°C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70°C and the knife at -23°C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.     

A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks

Diethylene glycol distearate is used as a removable embedding medium to produce embeddment -free sections for transmission electron microscopy. The easily cut sections of this material float and form ribbons in a water-filled knife trough and exhibit interference colors that aid in the selection of sections of equal thickness. The images obtained with embeddment -free sections are compared with those from the more conventional epoxy-embedded sections, and illustrate that embedding medium can obscure important biological structures, especially protein filament networks. The embeddment -free section methodology is well suited for morphological studies of cytoskeletal preparations obtained by extraction of cells with nonionic detergent in cytoskeletal stabilizing medium. The embeddment -free section also serves to bridge the very different images afforded by embedded sections and unembedded whole mounts.     

Photographic Mapping of a Tissue Surface to Locate Fields for Electron Microscopy; Mouse Lung

Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal stub and ultrathin sections cut from the faced tissue. The method is as equally suitable for the examination of other tissues, particularly when large areas and multiple sampling may be required.     

An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections.

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO(4), and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 +/- 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 +/- 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.     

A new technique for electron microscopic dry-mounting radioautography of soluble-compounds

Summary A new technique for dry-mounting electron microscopic radioautography of water soluble-compounds has been established.Tissues containing labelled compounds are cut as small as less than 1 mm in diameter, plunged into isopentane cooled to about –160° C with liquid nitrogen, and frozen-dried at –50° C for 24–48 hours. After drying, the tissues are embedded in Epon, which is polymerized according to the conventional procedure.Ultrathin sectioning is accomplished with ethylene glycol instead of water in the knife trough so that water is not involved in the floatation and expansion of the ultrathin sections. Sections are picked up on collodion coated grid meshes from ethylene glycol.Radioautographic emulsion is diluted 1 part in 10 parts with distilled water at 40° C. Ten ml of the diluted emulsion is added with 0.2 ml of 2 per cent aqueous solution of dioctyl sodium sulfosuccinate. A thin film of the emulsion is obtained by dipping a platinum wire loop into the emulsion. The loop is set on a flat surface of a desk for air-drying. The dried film is then applied to the mesh and it is warmed at 37° C for 1 hour in order to help the film to adhere to the mesh. Thus, dry-mounting of packed monolayers of radioautographic silver crystals can be constantly achieved. The mesh is then exposed, developed and stained according to the conventional technique.     

Improved Methods for Making and Using Glass Knives

We have observed over time that the right side of a glass knife is the optimal cutting edge for microtomy if the counterpiece (heel opposite the edge) is controlled within 1 mm. The right cutting edge has been considered the “saw toothed” side and has not been used for ultrathin sectioning. We have observed that the right cutting edge is sharper and more durable than the left. Light and scanning electron microscopy were used to observe the cutting edge, and transmission electron microscopy was used to examine semithin and ultrathin sections of animal and plant tissues cut by the right and left sides of the cutting edge. The results indicate that the cutting edge becomes sharper and more durable from left to right. Both the quality and efficiency of ultrathin sectioning is improved by using the right cutting edge.     

A Rapid Method for Resectioning 0.5-4 μ Epoxy Sections for Electron Microscopy

A simple and rapid method is described for resectioning semithin Epon sections which have been stained for light microscopy, mounted on slides, and examined under immersion oil. The immersion oil is removed with xylene and the section is air dried. A drop of distilled water is applied to the slide and a razor blade is slid under the section. Freed from the slide, the section floats on the surface of the water and is transferred to another drop of water on the surface of a smooth, newly prepared Epon block face. The water under the section is withdrawn with bibulous paper. The section is thoroughly dried and bonded to the block surface by briefly heating in a 60 C oven. The tissue may then be re-sectioned and stained for electron microscopy in the conventional manner. This method has been used by several different technicians to produce ultrathin sections equal in quality to those produced by conventional methods and it greatly facilitates the selection of critical areas for examination by electron microscopy.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

Sections from double mesas obtained at the same tissue plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3 degrees to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6 degrees in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

Summary The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.     

Accurate Placement of Ultrathin Sections on Grids; Control by Sol-Gel Phases of a Gelatin Flotation Fluid

A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.     

Electron microscopy of lung rapidly frozen under controlled physiological conditions

Light microscopy of lung rapidly frozen under controlled physiological conditions has been very successful in correlating pulmonary structure and function. However, to study some aspects of pulmonary capillary morphology, the higher resolution of electron microscopy (EM) is necessary. To date, most EM of lung has involed the instillation of a fixative through the airways or vascular system, techniques that probably alter the normal pressure relationships of the capillaries and therefore their morphology. We describe here a technique for rapidly freezing lung to a depth of 1--2 mm below the pleural surface and preparing sections for EM. Lungs from open-chest rats were frozen at various transpulmonary pressures with cold (--80 degrees C) 70% ethylene glycol. Small pieces were then fixed with a solution containing glutaraldehyde and paraformaldehyde for 24 h at --50 degrees C. Staining was with osmium tetroxide and uranyl acetate. Lung frozen at high volumes showed marked stretching of the alveolar septa with severe deformation of the capillaries. Lung frozen at low inflation pressures revealed open capillaries containing numerous red blood cells; in addition, infolding of the alveolar wall was frequently seen. We conclude that this technique gives a level of preservation of rapidly frozen lung suitable for electron microscopy.     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.