Sample preparation for electron microscopy of internal cell structure.

Methods are reviewed for examination of internal cell structure by high-resolution scanning electron microscopy and compared with the rapid-freeze deep-etch replica technique used in transmission electron microscopy. Rapid freezing of fresh material, followed by freeze-fracture, provides a theoretically attractive approach in ultrastructure studies, but the high protein and solute content of most cells prevents a deep three-dimensional view for material frozen without some form of extraction. After discussion of other methods it is concluded that the most useful general approach, at least for cultured cells, is to first permeabilize or break open the cells in a medium which preserves the structure under study in a functional state as, for example, the movement of chromosomes along the division spindle, or transport of proteins within the Golgi region. After permeabilization, with attendant partial extraction, the preparation can be fixed, then viewed by either deep-etch replication, or by high-resolution scanning electron microscopy, with structure of interest revealed in deep view.

Three-dimensional viewing of internal cell structure.

3T3 and HeLa cells, grown as a monolayer, have been rapidly frozen by propane jet as a fresh preparation, without pretreatment. In some experiments the frozen cells were fractured at -170 degrees C, thawed into fixative and viewed by high-resolution SEM after critical-point drying. In other experiments the frozen cells were thawed into fixative unfractured. These preparations were refrozen in 15% methanol, fractured and deep-etched for replication and TEM study. The technique used in this work appears to give rapid rewarming from -170 degrees C to 0 degree C with little evidence of ice crystal growth. The cells fractured before thawing, examined by SEM, show extensive extraction of both nucleus and cytoplasm with deep views of nuclear chromatin, and of cytoplasmic organelles caught amongst rather distorted filaments of the cytoskeleton. Initial fixation for the SEM work was light (0.3% glutaraldehyde for 10 mins) so that structure is seen as it would be retained for antibody labelling.

Freeze-fracture of 3T3 cells for high-resolution scanning electron microscopy.

Triton-extracted, freeze-fractured 3T3 cells have been examined in the Hitachi S-900 field-emission SEM, after light platinum coating, at low beam voltage to evaluate the performance of the microscope under these conditions. For unstained material fixed in glutaraldehyde alone, high-resolution images can be obtained, at accelerating voltages of 1.5-5kV, after rotary deposition of platinum to an average thickness of 1.5-3 nm. Comparisons are made between these results and those of studies by TEM of deep-etch replicas of similar material previously published.

Study of the conditions necessary for propane-jet freezing of fresh biological tissues without detectable ice formation.

The performance of a commercial double-propane-jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze-etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20-30 microns thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze-etch microtome within 5-10 microns of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.

Freeze fracture through the cytoskeleton, nucleus and nuclear matrix of lymphocytes studied by scanning electron microscopy.

The technique of delaying fixation until after freeze-fracture and thawing, described in an earlier paper (Haggis & Bond, 1979), has been developed further for study of cells in culture, principally mouse lymphocytes stimulated by concanavalin A. Using a thin layer of cells, a cryoprotectant concentration of either 10% glycerol or dimethylsulphoxide, is sufficient to give good structural preservation after rapid freezing and thawing. Nuclear matrices and Triton-permeabilized cells have been prepared from stimulated lymphocytes for comparative study. Polylysine-coated fibrin support films have been found to provide a convenient means of handling cells and subcellular preparations during freeze fracture, critical point drying and mounting for high-resolution scanning electron microscopy.

Contribution of scanning electron microscopy to viewing internal cell structure.

Freeze-fracture provides a way of opening up cells and tissues for an internal view of cytoplasm and nucleus, and can give an internal view of a cytoplasmic organelle if the plane of fracture cuts through the organelle. Selective removal of soluble or other components is necessary for a deep view of structure at the fracture face. This may be achieved by osmium digestion, or by glycerol extraction, or by delaying fixation until after freeze-fracture and thawing, or by prior treatment with detergent to remove cell membranes and wash out soluble components. Cells may also be ruptured at room temperature, or in certain cases prepared to expose the inner surface of the plasma membrane for scanning electron microscopy (SEM) viewing. The problems and potential of SEM viewing of the cell interior are in certain respects similar to those encountered and derived from TEM replica study of freeze-fractured cells after deep etching. TEM replicas give better resolution, while SEM offers advantages in study of surfaces with considerable depth of structure. Study of fresh material, without fixation or alcohol dehydration, by rapid freezing and deep etching, is increasing our understanding of the artifacts that can be produced by these two preparative steps which are essential to critical point drying, whether used as a preparative step for SEM or for whole-mount high-voltage TEM microscopy.

A new approach to the study of the E. coli nucleoid.

E. coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels. After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin. Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form. The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis. These spreads were critical-point dried, rotary shadowed with platinum-carbon and viewed as stereo-pair micrographs. Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines. After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form. Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure.

Three-dimensional view of the chromatin in freeze-fractured chicken erythrocyte nuclei.

The freeze-fracture thaw-fix (FfTF) technique described in earlier papers is applied in the present work to more detailed study of the chicken erythrocyte, by transmission replicas and high resolution scanning electron microscopy (3 nm scan beam size). The three-dimensional structure of the chromatin, and possibly the non-histone protein matrix, of fractured nuclei is to a large extent retained in this method of preparation and seen in stereomicrographs. In these micrographs the helical sub-structure of the 25 nm chromatin strands can be seen at about the same resolution as that of previously published micrographs in which extracted chromatin is viewed by negative contrast or after metal shadowing. The useful resolution of the secondary electron micrographs, for a suitably mounted specimen, is shown to be as good as that of transmission micrographs of platinum-carbon replicas of the same material.

Thrombus formation in a staged capillary membrane oxygenator associated with a microaggregate blood filter: a scanning electron microscope study.

Blood contact at interfaces in extracorporeal devices is a source of traumas. Proteins are very rapidly adsorbed; then, depending on which proteins are left various degrees of platelet aggregation follow and thrombi develop. The scanning electron microscope reveals very instructive information on the morphology of the blood deposits which adhere to foreign surfaces. Oxygenators such as the Awad D, which is a staged one made of silicone rubber, become "thrombus invaded" after prolonged extracorporeal circulation in spite of adequate heparinization of the blood. In most cases, evaluation of membranes and devices is assessed with respect to transfer of blood gases. Careful examination of the morphology of deposits should be developed. This study shows the importance of blood flow rate, design, and materials.

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.

Improved resolution in cathodoluminescent microscopy of biological material.

This paper continues work reported in an earlier paper on modification of a Cambridge Stereoscan Mk IIA to improve the quality of cathodoluminescent micrographs of biological material. In the work presently described the microscope gun has been offset laterally by 2 mm, to prevent light from the filament passing down through the column to the specimen chamber. The electron beam is brought onto the column axis by deflection coils. This modification effectively eliminates background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. Results for yeasts and wheat seed sections are described.

Intra-uterine devices, A SEM study on the Dalkon Shield.

A SEM study carried on intra-uterine devices (Dalkon Shield) is reported. The poor manufacturing of the devices is stressed, which could increase their efficiency but also the risks of penetration, or worse, perforation. The solution for the future could be copper bearing intra-uterine devices coated by hydrogels. This should maintain efficiency and improve security.

Preclotting of knitted Dacron prosthesis. A scanning electron microscope study.

A scanning electron microscope study of preclotting on knitted Dacron prosthesis is reported. Five steps of the interaction are well identified: (1) before any blood contact (virgin Dacron), (2) during the first 3 minutes (fibrin and platelet aggregates), (3) fifth minute of contact (clotting), (4) 15 minutes of contact with heparinized blood (thin fibrin network), and (5) the following minutes (invasion of fibrin, which enmeshes blood cells).

Nature of deposits in a tubular membrane oxygenator after prolonged extracorporeal circulation: a scanning electron microscope study.

Although silicone fibers are among the most compatible with tissue and blood, numerous deposits are observed after their prolonged usage in a capillary membrane oxygenator, even when the blood has been properly heparinized. The scanning electron microscope (SEM) study shows that the morphology of these deposits varies greatly, depending upon the part of the unit from which the sample is taken. The area close to the inlet is the most severely affected. The outlet zone is affected to a lesser degree, and the areas in between are only slightly affected.

Preclotting and thrombosis of a dacron prosthesis. A scanning electron microscope study.

The authors report eht study of preclotting made on a knitted Dacron prostheses before implantation into a patient. Within 24 h of implantation, clotting occurred. The patient had to be reoperated, and a second preclotting followed. Specimens were removed at different times and fixed in a Rembaum solution before being dried and studied by scanning electron microscopy.