A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy

Monolayers of PtK-1 and HeLa cells grown on glass or plastic supports are extremely susceptible to lacerations, e.g., splits and cracks caused mainly by shrinkage when prepared for scanning electron microscopy (SEM). We find that a four-step fixation procedure including glutaraldehyde, OsO₄, tannic acid, and uranylacetate application, in combination with critical point drying, drastically reduces these structural damages. In addition, the conductivity of the specimens is enhanced, so that they can be investigated without gold coating. Transmission electron microscopy (TEM) investigation of perpendicular sections in the area of lacerations provides evidence that the subcortical cytoskeletal elements are of crucial importance in maintaining cell membrane stability during the preparations. Our relatively quick and simple procedure results in an improved structural appearance of the cells.     

A preparation method for observing intracellular structures by scanning electron microscopy

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.     

The preparation of leaf surfaces for scanning electron microscopy: a comparative study

Cryopreservation is the superior technique for viewing leaf surfaces in the SEM. Epidermal cells become distorted when freeze dried and disrupt the orientation of epicuticular wax structures. The latter are largely lost during critical point drying. Nevertheless, the appearance of surface structures after subjecting them to each drying method is valuable in interpreting the features observed by cryopreservation.     

The preparation of human articular cartilage for scanning electron microscopy

This paper compares sixteen preparative techniques thought to be of advantage in the study by scanning electron microscopy (SEM) of human articular cartilage surfaces. The adequacy of surface preservation obtained with the techniques, was judged subjectively, first, by the reproducibility of secondary electron images of normal cartilage, and second, by comparing the results with those obtained by reflected light microscopy of the fresh unfixed cartilage surface over a magnification range of × 20 – × 240. Adequate surface preservation was confirmed when cartilage surfaces had been dehydrated through ethanol to propylene oxide and vacuum dried; dehydrated through amyl acetate and quenched in Freon before freeze-drying; dehydrated and passed through amyl acetate at low temperature before freeze drying. Valuable information can be obtained from different specimens by varying the technique of preparation. At different ages, different surface features are best preserved. In a systematic study it has been found essential to adopt a uniform preparative method and to control the results by reflected light microscopy. Even with the most perfect preparation, the surface appearances cannot be identical with those that function under load in vivo.     

Freeze-fracture for scanning electron microscopy

Two different freeze-fracture methods are explored for preparation of biological material for scanning electron microscopy. In the simpler method the tissues are first fixed and dehydrated. They are then frozen and fractured, and after thawing, critical-point dried. This method has already been used in a number of studies of animal tissues (heart, liver, kidney). It is applied here to the examination of plant material (leaf mesophyll cells). In the second method tissues, or cells, are first infiltrated with cryoprotectant (dimethylsulphoxide) then frozen and fractured, and not fixed until after thawing. The fixed tissues are finally dehydrated and critical-point dried. This method also has previously been used in the study of animal tissues, and is applied here to carrot protoplasts, chicken erythrocytes, and leaf mesophyll cells.     

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.     

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 method for the removal of epoxy resins from tissue in preparation for scanning electron microscopy.

A method for unmounting entire resin-embedded samples for SEM observation is described. This technique is particularly useful when correlation of TEM and SEM images is desired for material that is no longer available for conventional SEM preparative procedures. Sample embedded in a variety of epoxy-type resins were trimmed of excess resin and placed in a concentrated solution of sodium methoxide. After complete dissolution of the resin, the tissue was washed in a graded series of sodium methoxide in methanol--benzene, transferred to acetone, critical point dried, mounted on stubs, and coated with gold-palladium. Upon viewing in the SEM, the tissue sample showed remarkable preservation of detail at relatively high magnifications.     

Dinosaur eggshell study using scanning electron microscopy

Visualization and analysis of structural features in fossil dinosaur eggs by scanning electron microscopy augment information from traditional petrographic light microscopy. Comparison of characteristics in fossil and modern eggshells allows inferences to be made regarding dinosaur reproductive biology, physiology, and evolutionary relationships. Assessment of diagenetic alteration of primary eggshell calcite structure that occurs during fossilization provides important information necessary for taxonomic identification and paleoenvironmental interpretations.     

A specimen holder for low-temperature scanning electron microscopy

A miniature vise built into a copper stub is described that holds bulk, pre-frozen, hydrated biological specimens during examination under the electron beam of the scanning electron microscope.     

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.     

A method for observing intracellular structures of free cells by scanning electron microscopy

A new method is described for scanning electron microscopic observation of cultured free cells using chitosan embedding. Intracellular structures of free cells can be clearly observed using a combination of the chitosan embedding and the Osmium–DMSO–Osmium (O–D–O) method. Chitosan, a polysaccharide, is not affected by the osmium maceration procedure central to the O–D–O method, which destroys other possible embedding media such as gelatin, agar, egg albumin and fibrin. Chitosan cracks to yield a fracture face which is featureless at low magnifications, and causes no recognizable artefacts.     

Problems with the preparation of blood vessels for scanning electron microscopy a critical review

The application of the scanning electron microscope (SEM) in the investigation of blood vessels in the last ten years is reviewed. Methodological differences from anesthesia of the experimental animal to mounting of the dried tissue specimen are discussed. Some of the major interpretative pitfalls are indicated and some practical guidelines for standardization are offered.