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.

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.

Some years ago we introduced a thaw-fix technique for SEM viewing of internal cell structure1. In thisptechnique cells or tissues are frozen unfixed, fractured at -170 to -190°C and thawed into fixative at room temperature. In early work we infiltrated cells with glycerol or dimethylsulphoxide (to 25-30%) prior to freezing, to avoid ice crystal artifact during freezing and thawing. This infiltration took 20 mins, to 1 hr. with the possibility of ultrastuctural change during this time, since the cells were unfixed. Results for interphase chromatin were interesting but cytoplasmic structure was not well preserved. We have now improved our techniques for rapid freezing and rapid thawing and report results here for fresh material, rapidly frozen without cryoprotectant, showing excellent cytoplasmic preservation. Interphase chromatin shows structural detail comparable to that obtained earlier with glycerol cryoprotection.3T3 cells were grown on 20μm fibrin films, rapidly frozen by propane-jet freezing, fractured in a Balzers BAF 400 freeze-etch unit at -170°C and thawed into a fixative of Bershadsky buffer3 + 1.5% glutaraldehyde.

SUMMARYTriton‐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 1.5‐3nm. Comparisons are made between these results and those of studies by TEM of deep‐etch replicas of similar material previously published.

The results reported in this paper form a direct extension of the work of Heuser and Kirschner. These authors permeabilized cultured fibroblasts with Triton X-100 prior to rapid freezing, freeze-drying and rotary platinum coating for TEM viewing. We have permeabi1ized 3T3 cells in a stabilization buffer similar to that used by Heuser and Kirschner, rapidly frozen and fractured the permeabilized cells, rotary coated the preparations with platinum after both deep etching and critical-point drying and viewed them by both TEM and high-resolution SEM. By fracturing the cells we obtain an internal view of the nucleus as well as of the cytoplasm.The 3T3 cells were cultured on a film of fibrin 20 μm thick (for reasons described below). The cells were permeabi1ized at room temperature for 15 min with 0.5% Triton X-100 in Bershadsky buffer (50 mM imidazole 50 mM KC1, 0.5 mM MgCl2, 1 mM EGTA, 0.1 mM EDTA, 1 mM mercaptoethanol, 4M glycerol at pH 6.7).

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.

SUMMARY Experiments were carried out to determine whether propane‐jet freezing could be as satisfactory as impact freezing in deep etch work. The material used was the intestinal brush border, previously studied by Heuser and coworkers where the 5–6 nm decoration of actin rootlet filaments, and the fine network of filaments linking these rootlets, provide good criteria by which to judge the quality of the preparation, as regards ice crystal growth and surface contamination. Propane jet freezing was indeed found satisfactory provided appropriate conditions were met (viz thin specimen, fracture near the surface). Variable results were obtained until it was realized that with a Balzers freeze‐etch unit fitted with a rotating specimen table there is a 10–15 min delay (when specimen temperature is reset) between the time the recording thermocouple shows a given temperature to have been obtained and the time the specimen block actually reaches this temperature. Appropriate allowance must be made for this lag to achieve satisfactory deep etch replicas.

SUMMARYThe 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.

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.

SUMMARYE. 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.

SUMMARY Vicia faba root slices have been frozen, fractured and thawed into fixative, to give a deep view into cell cytoplasm and into cell organelles. Fracture through interphase nuclei shows a tangle of 20–25 nm chromatin as well as finer strands. The nucleoli appear very compact. The proplastids in these preparations are packed with starch grains. In fracture through smaller organelles, probably mitochondria, 10–12 nm strands are seen which may correspond to organelle DNA in supercoiled form. Strands of this dimension, as well as finer strands, are also seen in preparations of isolated leaf chloroplasts after freeze‐thaw lysis.

SUMMARY 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.

We have previously described a method for three-dimensionally viewing the interior of cells by SEM. Thin slices of tissue are used, or alternatively, cells in suspension are embedded in a thin layer of fibrin gel. The gels or tissue slices are infiltrated with glycerol or dimethyl sulphoxide (DMSO) to suitable concentration, rapidly frozen, fractured, and thawed into fixative solution. The fracture surface is viewed after critical point drying and gold, or gold-palladium, coating. In the present report we extend this work in two ways, first to show how varying conditions at the moment of thawing can be used to modify the structure seen, creating a miniature in vitro control system for study of the cell interior, and secondly to show that replicas from the fracture surface can be obtained for examination by TEM. Using replicas, this technique becomes an extension of the standard freeze-etch technique, but with a result equivalent to very deep etching.

SUMMARYTwo 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.

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.

Scanning electron microscope studies of anthers of the dicotyledons Helianthus annuus, Solanum tuberosum, and Lupinus polyphyllus, and the monocotyledons Iris pseudo-acoris and a Lilium hybrid revealed discontinuities or holes in the meiocyte callose wall and continuity of the plasma membranes of adjacent meiocytes. The holes in the callose wall generally were confined to areas where neighboring meiocytes were in contact. The holes varied in size within locations and between taxa. The largest holes, about 2.4 μm diameter, were found in Lupinus. Fixation in standard acid–alcohol fixatives resulted in marked plasmolysis and loss of cytoplasmic detail, but the nucleolus and bivalents were readily apparent. Fixation in buffered glutaraldehyde, with or without postfixation in OSU4, preserved the cytoplasmic organelles and plasmolysis was minimal, but bivalents could not be distinguished. All fixatives preserved the nuclear membrane so that the nuclear region was clearly delimited from the cytoplasm.

Scanning electron microscope studies of anthers of the Asiatic Lilium hybrid Enchantment and Solanum tuberosum L. cv. Netted Gem fixed in organic acid – alcohol type fixatives clearly revealed nucleoli, bivalents, and the meiotic spindle. Centromere regions could not be identified in pachytene bivalents, but areas of possible spindle attachment were evident for metaphase I – anaphase I bivalents.

SUMMARYMethods are described for the use of a sensitive photomultiplier, improved photomultiplier voltage supply circuitry, quartz lens and a half‐parabolic mirror to increase the amount of light collected from a luminescing specimen examined in the scanning electron microscope. The combined effect of these improvements increases cathodoluminescence detection twenty times. Sources of background light are investigated and recommendations are made for decreasing this noise component, which seriously limits sensitivity of cathodoluminescence signal detection.An appendix describes the natural luminescence (autoluminescence) of plant tissues under the impact of the electron beam. The biological applications of this mode of scanning microscopy have been little explored, but autocathodoluminescence is found to be of wide occurrence in plant tissues.

The fine structure of the myo-cuticular junction in an acarid mite, Caloglyphus mycophagus, is described. The muscle fibres are attached to the cuticle via flattened, much invaginated, epidermal cells. Unlike the situation described for other arthropods, the stress across these epidermal cells does not appear to be transmitted by microtubules but rather by desmosome-like structures which form intraepidermal cell bridges where invaginations from the outer and inner surfaces of the epidermal cells lie close together. The muscles are attached to the inner surface of this complex desmosome and the outer surface is linked to the cuticle by extracellular fibrils.