ADVANTAGES AND APPLICATIONS OF LOW TEMPERATURE SCANNING ELECTRON MICROSCOPY WILLIAM P. WERGIN Electron Microscopy Laboratory, Agricultural Research Service Beltsville, Md. 20705 USA ROBERT W. YAKLICH Soybean and Alfalfa Research Laboratory Beltsville, Md. 20705 USA and ERIC F. ERBE Electron Microscopy Laboratory Beltsville, Md. 20705 USA ABSTRACT Low temperature scanning electron microscopy (LTSEM), which enables observations of frozen hydrated samples, permits imaging of some specimens that cannot be processed or properly preserved for ambient temperature SEM studies. LTSEM specimen preparation merely consists of cryofixation; therefore, artifacts arising from chemical fixation, solvent dehydration and critical point drying are averted. The cryotechnique is particularly useful to observe specimens that are: 1) soluble in aqueous fixatives or dehydrating solvents; 2) impermeable to chemical fixatives; 3) delicate and thereby subject to mechanical damage; 4) loosely associated or easily dislodged or; 5) physiologically altered by either the water or the time that is required for conventional chemical fixation. LTSEM also allows comparisons of freeze-fractured and freeze-etched faces of a specimen; this advantage can be used to determine the location of the water in the specimen and to distinguish aqueous areas from naturally occurring air pockets and non etchable solids. In the past, the resolution of frozen hydrated samples was limited by the conventional SEM; however use of cryotechniques on a field emission SEM enables imaging of macromolecular structures such as membrane particles, which measure less than 10 nm. 1.Introduction For many biological specimens, conventional scanning electron microscopy (SEM), which is performed at ambient temperatures, generally requires preparation procedures that consist of chemical fixation, dehydration, critical point drying and coating. Each of these procedures has been associated with artifacts or structural changes in the sample (Beckett and Read, 1986; Read and Jeffree, 1988; Wergin and Erbe, 1989; Jeffree and Read, 1991; Read, 1991; Read and Jeffree, 1991, Echlin, 1992). For example, chemical fixation, which requires relatively long periods in an aqueous fixative (5-60 min), may fail to stabilize all structural components and may actually dissolve some materials; solvent dehydration may also extract cellular constituents such as lipids and waxes; critical point drying can shrink tissue as much as 30% and sputter coating with 20-30 nm of a heavy metal will obliterate many fine structural details. To avoid these artifacts, examination of hydrated tissues would be desirable. Unfortunately, because specimens that are examined with the SEM must be placed in a vacuum, the surface tension resulting from evaporation of water, which frequently constitutes as much as 90% of the total tissue weight, would quickly result in collapse and distortion of the specimens. About 25 years ago, Echlin (Echlin et al., 1970; Echlin, 1978) described a procedure that would avoid these problems. This procedure, which has become known as low temperature (LT) SEM allows examination of fresh, frozen, fully hydrated specimens. Refinements in the procedure, which were made by Pawley and Norton (1978) and Robards and Crosby (1979), led to the introduction of commercial low temperature systems designed for use with a conventional SEM. Use of LTSEM has several distinct advantages. Compared to chemical fixation, the time required for cryofixation is relatively short. Although the freezing rates for biological tissues may vary depending on the size and nature of the specimen, the time necessary to quench freeze a sample is shorter, by several orders of magnitude, than that required for chemical fixation (Bachmann and Talmon, 1984). In addition, the specimen is not subjected to any drying conditions. Therefore, forces associated with surface tension do not collapse or distort the tissue. The frozen hydrated specimen is actually observed on a cryostage that operates at temperatures below -130 C; consequently the vapor pressure of water is not significant and sublimation does not occur at a detectible rate. Furthermore, at temperatures, below 130 C, recrystallization of pure water-ice does not occur (Beckett and Read, 1986). Consequently, as long as the stage remains cold, a frozen sample remains stable and can be observed for several hours (Wergin and Erbe, 1989; 1991a; 1992). In spite of these advantages, LTSEM has several disadvantages. Namely, frozen specimens are more difficult to manipulate and mount, they are more prone to surface contamination from condensates, the resolution of the conventional technique is generally limited to magnifications below 10,000X and frozen samples are more difficult to store. In 1986, Nagatani and Saito (1986) introduced the field emission (FE) SEM, which has a resolution of less than 1 nm, and Mller et al. (1986; 1990; 1991) described a new generation of cryochamber, which were designed for high vacuum use and incorporate planar magnetron sputter coating. These two developments have revitalized biological interest in LTSEM. This paper illustrates and discusses some of the advantages and applications of low temperature scanning electron microscopy that we have experienced in our laboratory. 2.Materials and Methods 2.1 Bulk Specimens The low temperature manipulations and observations in our laboratory were obtained either with an Oxford CT 1500HF Cryotrans System that was installed on a Hitachi S-4100 FESEM or with an EMscope SP2000A that was mounted on a Hitachi S-570 SEM. Details of the preparation procedures and the modified specimen holders that were used in this study have been previously described (Wergin and Erbe, 1989; 1991a). Briefly, specimens were mounted with Tissue TEK either on standard Oxford or EMscope specimen holders or on modified holders that could be accommodated by the commercial units. The holders containing the specimens were plunge-frozen in liquid nitrogen slush and cryotransferred to a prechamber where they were sputter coated with gold or platinum then inserted onto the cryostage of the microscope. 2.1 Fractured Specimens For observing internal structures, the frozen specimens were transferred to the prechambers where they were fractured with a pick, knife, or hinged specimen holder, etched by raising the temperature to -950C, coated as described above and moved to the cryostage in the microscope for observation. To compare freeze-fractured and freeze-etched faces in the same specimen, the samples were frozen, fractured and etched in the prechamber. Next, a scalpel blade was used to refracture one-half of the sample. Then the entire sample was coated and inserted onto the cold stage of the microscope and the area where the freeze-fractured face intersected with the freeze- etched face was located. All samples were observed with accelerating voltages of 1 to 10 kV and images were recorded onto Polaroid Type 55 P/N film. 3.Advantages and Applications 3.1 Soluble Samples Many types of samples that are prepared for SEM examination commonly contain 75 to 95% water. These highly hydrated samples not only include experimental biological specimens but also many natural, processed and frozen foods and food products. In addition, these samples as well as various nonbiologicals, such as pharmaceuticals and petroleum products, contain lipids, carbohydrates, such as starch and waxes, and other materials that would be soluble in the aqueous fixatives or organic solvents generally used to fix and dehydrate samples for conventional SEM. LTSEM, which employs cryofixation, freezes and preserves these soluble materials. Snowflakes represent the ultimate examples of specimens that would be soluble in a chemical fixative. A single snowflake consists of an aggregation of subunits commonly referred to as snow crystals. The snow crystals illustrated in Figure 1 largely consist of hexagonal plates. However, snow crystals exhibit an infinite variety of shapes that include columnar, needle, plate-like and the more familiar hexagonal dendritic forms, one of which is illustrated in Figure 2. Aside from a microscopic nucleating particle, which initiates the formation, each snow crystal generally consists of a single ice crystal. Although replicas of snow crystals have been made and examined in a transmission electron microscope (TEM) (Stoyanova et al., 1987), cryofixation and LTSEM have enabled observation and description of the various natural forms of frozen, precipitated snow crystals (Wergin and Erbe, 1994; Wergin et al., 1995). Although snow crystals represent an extreme example of a soluble sample, plants, animals and insects frequently exude secretions or contain surface coatings that would be either dissolved or removed from the surface of the specimen during conventional SEM preparation. Figure 3 illustrates a portion of the stigma from a Cosmos flower. In plants, the stigma consists of glandular tissue that secretes materials which help entrap and promote germination of the pollen grains. In Cosmos, the epidermal cells of the stigma consist of the elongated papillae. The pollen grains are embedded in the material secreted by these cells. 3.2 "Unfixable" Material Some biological specimens contain a covering or layer of cells that is nearly impermeable to the aqueous fixatives and dehydration agents normally used for conventional SEM preparation. Numerous examples include dry seeds, fungal spores, pollen grains and insects. Figure 4 illustrates an egg from the nematode Meloidogyne incognita. These nematode eggs, which are designed to resist environmental fluctuations in the soil, commonly measure 40 by 90 mm. Although their small size would generally enhance good chemical fixation, the egg shell, which consists of a chitinous layer, is fairly impervious to chemical fixatives. Consequently fixation in glutaraldehyde, followed by dehydration, freeze-fracturing and critical point drying for conventional SEM fails to resolve any significant structural information about the developing embryo (Fig. 5). The contents, which appear granular and fibrillar, have no structural significance. Alternatively, cryofixation and freeze-fracture of these nematode eggs preserves the structure of developing embryos (Fig. 6), has been used to describe embryogenesis of the nematode from the single cell stage through the development of the first stage larvae (Orion et al., 1994). 3.3 Loosely Associated Many biological problems that one desires to study with an SEM involve two or more structures or organisms that are only tenuously associated. Submerging these complexes into chemical fixatives and processing them for conventional SEM observation would either disturb or alter such an association. LTSEM can frequently be used to image these precarious associations. Figure 7 illustrates an instar of the sweet potato white fly that is feeding on a tomato leaf. Chemical fixation of this tissue, which requires several minutes, causes the instar to cease feeding and dislodge from the surface of the leaf. As a result, observation of the leaf at ambient temperature in a conventional SEM would fail to identify the nature of the insect problem. In Figure 8 a single pollen grain is precariously situated between the columnar epidermal cells of the stigma from common ragweed (Ambrosia artemisiifolia L.). At this early stage of pollination, no obvious secretions, which would help anchor the pollen grain, are apparent. This relationship would not be retained during conventional tissue processing. More complex tissues containing loosely affiliated internal structures are also maintained by cryopreparation procedures. Figure 9 illustrates an anther that has been cryofractured to reveal the internal locules. Mature pollen grains can be observed within the locules as well as on the surface of the anther. Unfortunately the mechanical trauma resulting from the fracturing process can also produce artifacts as evidenced by the presence of the pollen grains on the fractured surface of the anther wall. 3.4 Delicate Structures Delicate specimens or those with fragile appendages frequently suffer from mechanical damage during conventional SEM preparation. The damage may result from the turbulence that occurs when samples are transferred from fixatives, rinses and dehydrating solutions or from trauma during the critical point drying procedure which is required for conventional SEM preparation. Mechanical damage to external structures is rarely observed in cryofixed specimens. Figure 10 illustrates fungal conidiophores (Erysiphe cichoacearum) consisting of a chain of single cells, about 150 mm in length, that arise from the surface of a sow thistle leaf (Sonchus arvensis L.). These fragile structures show no sign of mechanical damage, nor do the cells exhibit the wrinkled or shrunken appearance that characterizes many fungal hyphae that are prepared for conventional SEM. Similarly, Figure 11 illustrates the well preserved setae, 200 to 300 mm in length, that arise from the surface of a gypsy moth larvae (Porthetria dispar). The hooked tips of two trichomes from a bean leaf (Phaseolus vulgaris) are shown in Figure 12. Although these configurations are generally maintained in tissues that are cryofixed for LTSEM examination, they have not been encountered in leaf tissue that was processed by conventional procedures. 3.5 Physiologically Active Complete chemical fixation may require 30 minutes or more for some tissues. During this time many physiological and structural changes can occur in the tissue that is being processed. For example, nematodes generally require extended fixation schedules. The vinegar eel worm has actually been observed alive in a 3% glutaraldehyde solution after 24 hrs (Wergin, unpublished). During chemical fixation, nematode larvae gradually relax, loose their sinuous shapes and assume very straight configurations. Alternatively, the natural sinuous shapes of nematodes exhibited by living larvae are nicely preserved by cryofixation, which probably occurs in microseconds (Fig. 13). Plant physiologists have long been interested in guard cells, which are generally found on the lower surfaces of leaves. Changes in turgor pressure of these cells regulate the opening and closing of the stomata and thereby regulate air exchange between the inner tissues of the leaf and the environment. The guard cells readily respond to changes in moisture, temperature, light conditions, pollutants as well as other environmental factors. Investigators have frequently attempted to determine whether an adverse treatment influenced the guard cells by chemically fixing the treated leaves and determining whether the stomates were opened or closed when viewed by conventional SEM procedures. Unfortunately, chemical fixation by itself will affect the status of the stomates; therefore cryofixation and LTSEM are much more reliable for determining the condition of stomates following physiological treatment (Fig. 14). In addition, other features of the leaf, such as the delicate wax platelets on the surface of the epidermis are well preserved with cryofixation. The degree of hydration in some specimens will be affected by aqueous fixatives. For example, many plant materials may contain 85-90% water; however, as seeds develop and mature the moisture level is gradually reduced to as little as 10%; whereas, during subsequent germination, the moisture level gradually returns to 85-90%. Attempts to examine the different stages of seed development and germination by using conventional SEM or TEM would be compromised by the aqueous fixatives which would alter the degree of hydration in the seed tissues. Alternatively, cryofixation does not affect the moisture level of the sample (Fig. 15). 3.6 Freeze-Fractured/Freeze-Etched Faces Cryofixation followed by freeze-fracturing results in relatively smooth surfaces that exhibit few structural details in the LTSEM. To solve this problem a technique known as freeze-etching was initially developed by Steere (1957) for TEM observations of virus particles. The technique, which sublimes water-ice from the fractured surface of a sample, results in surface topography due to the exposed structural components on the freeze-etched face. This technique was further enhanced by recovering both halves or complementary pairs of a fractured sample, etching one of the surfaces and then comparing the complementary replicas from the freeze-fractured and freeze- etched images in the TEM (Steere and Erbe, 1979). Recently, similar techniques were used on frozen, hydrated samples to examine complementary halves of freeze-fractured, freeze-etched specimens by LTSEM (Wergin and Erbe, 1992). Imaging the complementary pairs of frozen, hydrated specimens in the SEM is faster than imaging complementary replicas in the TEM. However, the procedure requires specialized holders and is technically demanding. To simplify comparisons of freeze-fracture, freeze-etch images, samples can be frozen, fractured and etched in the prechamber of the cryosystem. Next, a scalpel blade is used to refracture one-half of the sample. Then the entire sample is coated and inserted onto the cold stage of the microscope. By locating the area where the freeze-fractured and freeze-etched faces intersect, images of both faces can be compared in the same field of view. Comparisons of the two images from a hot dog (Fig. 16) and pizza dough (Fig. 17) quickly identify the relative location and the extent of hydration in the sample and distinguish the free water from gas spaces and other nonetchable components such as lipid droplets and protein bodies. Comparisons of the two faces obtained from a cherry tree twig (Figs. 18) indicate that under winter conditions free water is generally confined to thin layers around the plasma membrane, tonoplast and other cellular organelles. The vacuoles appear to be cryoprotected by natural sugars. These results demonstrate that a simple procedure can be used to distinguish water from gas spaces and other nonetchable solids in a sample. The location of free water in a sample can be visualized. In addition the procedure can be used to determine the effects of cryoprotectants, to ascertain the affects of preparation variables such as various cryogens and to determine the ideal times and temperatures required for optimum etching. 3.7 High Resolution Conventional SEMs equipped with cryosystems rarely produce satisfactory images of specimens at magnifications above 10,000x. Reintroduction of the field emission SEM in 1986 (Nagatani and Saito, 1986), along with the development and integration of high vacuum cryosystems and magnetron sputter coating, which increased resolution on frozen tissues to less than 10 nm, provided a new high resolution technique for imaging frozen hydrated specimens. Although the general shapes of snow crystals could be easily determined with a conventional LTSEM, details about the surface structures remained elusive. Use of field emission LTSEM is now being used to illustrates the symmetrical microchannels that occur on the surface of crystals. Figure 19 illustrates a portion of a single arm of a dendritic snow crystal containing portions of two channels (left) and two depressions (right) that are less than 10 mm in width. In biological tissue, the resolution provided by the field emission LTSEM allowed investigators to image nuclear pores, 75 to 100 nm in diameter, on the surface of a fractured nuclear membranes from yeast cells (Figure 20). Finally, this combination of instrumentation and techniques was able to illustrate macromolecular structures in frozen hydrated biological material. The freeze-fractured image of the plasma membrane from a frozen hydrated yeast cell shown in Figure 21 was recorded at 100,000x and illustrates the hexagonal arrays of membrane particles that are characteristic of the P-face of this membrane. The particles, which probably represent protein/lipid complexes, have a diameter of about 10nm. Imaging membrane particles, which requires a resolution of less than 10 nm, was previously only possible on replicas that were viewed in a TEM. Within the last few years, several groups of investigators (Mller et al., 1990; Walther et al., 1990; Wergin and Erbe, 1991a; 1991b; Walther et al., 1995) have used different types of field emission SEMs, cryosystems and coating techniques to attain membrane particle resolution in frozen hydrated yeast cells. 4.Summary Introduction of the field emission scanning electron microscope as well as new cryosystems and coating techniques have resulted in revitalized interest in LTSEM. Many samples that can not be readily examined under ambient temperature in an SEM can now be imaged in their frozen-hydrated state using this technique. Specimen preparation for LTSEM merely utilizes cryofixation and thereby avoids the conventional processes consisting of chemical fixation, solvent dehydration and critical point drying, which frequently cause artifacts such as solubilization, shrinkage and distortion in many specimens. In addition, LTSEM enables observation of many types of specimens that could not be easily examined in an ambient temperature SEM. These specimens include materials that are soluble, unfixable, loosely attached or delicate. Observation of freeze-fractured and freeze-etched faces of a sample enable the investigator to distinguish spaces that are normally occupied by water from those that are occupied by gases. In addition, both of these components can be distinguished from the nonetchable solids that may also be present in the sample. Finally the field emission SEM properly equipped with a cryosystem enables investigators to observe macromolecular particles less than 10 nm in diameter on frozen, hydrated samples such as the membrane particles that exist on the plasmalemma. In the past, these structures could only be observed in replicas that were viewed in a TEM. Unfortunately LTSEM is not a panacea. The cryosystems, which cost as much as s $100,000, require additional training, expertise and expense to maintain and operate. Storage of frozen samples, which must be maintained in liquid nitrogen, requires careful handling and adds expenses, which are not significant considerations with ambient temperature samples. Manipulation of frozen samples is more tedious and must be done with care to avoid contaminants that will readily condense onto the surface of a frozen sample. Probably the most important problem now facing low temperature investigations is reducing the size of ice crystals that occur during the freezing process. Because most biological samples contain 75 - 95% water, formation of ice crystals can significantly disrupt the internal structure of cells. Much research is being done to reduce and avoid this problem. High pressure freezing, use of cryoprotectants, and cryogens other than liquid nitrogen are being evaluated for use in LTSEM studies. Future advancements in LTSEM will largely result from scientific advancements in these areas. 5.Acknowledgements The authors thank Christopher Pooley for converting the SEM negatives to the digital images that were used to illustrate this study. 6.References 1. Bachmann L, Y Talmon. 1984. Cryomicroscopy of liquid and semiliquid specimens: Direct imaging versus replication. Ultramicroscopy 14: 211-218. 2. Beckett A, ND Read. 1986. Low-temperature scanning electron microscopy. 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Using high-vacuum evaporation to obtain high resolution low-temperature images of freeze- fractured membranes from yeast. Proc. Electron Microsc. Soc. 49: 514-515. 25. Wergin WP, EF Erbe. 1992. Techniques for obtaining and observing complementary images with a low-temperature field emission SEM and subsequent comparison of the identical cells in freeze-etch replicas viewed with a TEM. Scanning 14: 17-30. 26. Wergin WP, EF Erbe. 1994. Can you image a snowflake with an SEM? Certainly! Proc. Royal Microsc. Soc. 29: 138-140 27. Wergin WP, A Rango, EF Erbe. 1995. Observations of snow crystals using low-temperature scanning electron microscopy. Scanning 17: 41-49 Figures were recorded on a Hitachi S-4100 field emission SEM equipped with an Oxford CT 1500 HF Cryotrans System unless stated otherwise. Figure 1. Snowflake composed of an aggregation of snow crystals that largely consist of hexagonal plates. The specimen was obtained from newly precipitated snow that was collected at Beltsville MD. Bar = 1.0 mm. Figure 2. Snow crystal from newly precipitated snow that was collected at Bearden Mountain, West Virginia. This crystal is a dendritic form that contains a central hexagonal plate. Minute ridges and grooves can be observed on the surface of the arms of the dendritic crystal. Bar = 500 mm. Figure 3. A portion of the stigma from a Cosmos sp. flower. The epidermis consists of glandular tissue that secretes materials which help entrap and promote germination of the pollen grains. The pollen grains are embedded in the material secreted by these cells. Bar = 100 mm. Figure 4. Single egg, measuring 35 mm by 75mm, from the nematode, Meloidogyne incognita. The egg shell consists of a chitinous layer that is not readily permeable to chemical fixatives. Bar = 30 mm. Figure 5. Eggs from the nematode, Meloidogyne incognita, that had been chemically fixed, dehydrated, cryofractured and critical point dried for observation at ambient temperature in a conventional Hitachi S-530 SEM. The internal tissues in these developing nematodes were not preserved; no significant structural information can be obtained from this preparation procedure. Bar = 50 mm. Figure 6. A group of nematode eggs from Meloidogyne incognita that had been cryofixed, fractured and imaged with the low temperature SEM. This preparation procedure preserved the internal structure of the developing nematodes and allowed one to identify the various stages of embryogenesis. Bar = 60 mm. Figure 7. Sweet potato white fly instar on the surface of a tomato (Lycopersicon esculentum) leaf. Tomato contains two types of trichomes, a multicellular tapered hair and a glandular form. Bar = 150mm. Figure 8. Single pollen grain that lies between the columnar epidermal cells of the stigma on a flower from common ragweed (Ambrosia artemisiifolia L.). This association may represent an early stage of pollination. No cellular secretions, which would help retain this association, are apparent. Specimen was imaged on a Hitachi S-570 SEM equipped with an EMscope SP2000A cryosystem. Bar = 20 mm. Figure 9. Cross fracture through an anther from wild columbine (Aquilegia canadensis). Mature pollen grains can be observed in the locules of the anther as well as on the external and fractured surfaces. Bar = 100 mm. Figure 10. Conidiophores of a powdery mildew fungus (Erysiphe cichoacearum) arising from the surface of a sow thistle leaf (Sonchus arvensis L.). Bar = 100 mm. Figure 11. Surface setae, 200 to 300 mm in length, that emanate from the surface of a gypsy moth caterpillar (Porthetria dispar). Specimen was imaged on a Hitachi S-570 SEM equipped with an EMscope SP2000A cryosystem. Bar = 100 mm. Figure 12. "Hooked" ends of single celled trichomes from the leaf surface of a pinto bean (Phaseolus vulgaris). Specimen was imaged on a Hitachi S-570 SEM equipped with an EMscope SP2000A cryosystem. Bar = 15 mm. Figure 13. Larval forms of the nematode, Pratylenchus penetrans. Cryofixation preserves the sinuous shapes that these nematodes assume in the living state. Bar = 150 mm. Figure 14. Lower surface of a soybean leaf showing guard cells and tightly closed stomata. The surface of the epidermal cells also exhibits wax platelets, which tend to be lost during conventional fixation procedures. Specimen was imaged on a Hitachi S-570 SEM equipped with an EMscope SP2000A cryosystem. Bar = 20 mm. Figure 15. Several cells from a cotton (Gossipium hirsutum) radicle that had been excised from a developing seed. Cryofixation, which does not alter the degree of hydration in this tissue, preserves structural features, such as the nucleus and nucleolus that are evident in the central cell. Specimen was imaged on a Hitachi S-570 SEM equipped with an EMscope SP2000A cryosystem. Bar = 15 mm. Figure 16. Rising pizza dough that was freeze-fractured (upper half of micrograph) and freeze-etched (lower half) to illustrate the distribution of water. The "holes" shown in the lower portion of the micrograph illustrate the distribution of the water that was etched from the fractured sample. Bar = 30 mm. Figure 17. Portion of an all beef hot dog that was freeze- fractured (upper half of micrograph) and freeze-etched (lower half). "Holes" indicate that the water was fairly evenly distributed throughout the sample. Large spherical structures that were not affected by the etching process probably represent fat droplets. Bar = 15 mm. Figure 18. Portion of woody tissue from a cherry twig. Left part of the micrograph was freeze-fractured and the right portion was freeze-etched. Etching reveals that the free water tends to be localized around the membranes. The vacuole, which was not affected by the etching, appears to be naturally cryoprotected. Bar = 20 mm. Figure 19. Portion of a newly precipitated snow crystal that was collected in West Virginia. Portions of two symmetrical channels (left) and two small indentations (right) that can be resolved on the surface of an arm from a dendritic snow crystal. Bar = 15 mm. Figure 20. Portion of the fractured yeast cell illustrating the membranes of the nucleus. This sample was fractured and coated with Pt/C in a high vacuum evaporator and then transferred to the stage for LTSEM observation. Nuclear pores, about 100 nm in diameter, can be seen on the surface of the nuclear membrane. Bar = 750 nm. Figure 21. Portion of the fractured plasma membrane from a yeast cell. The hexagonal arrays of membrane particles can be resolved on the fractured P-face of the membrane. These particles are about 10 nm in diameter. Bar = 300 nm.