Journal of Microscopy, Vol. 168, Pt 2, November 1992, pp. 169-180. Received 15 November 1991; revised and accepted 1 July 1992
High-resolution scanning electron microscopy of frozen-hydrated cells by P A U LWALTHER, YA CHEN,L O U I S L. PECH* and J A M E S B. P A W L E Y , Integrated Microscopy Resource, 1675 Observatory Dr., Madison, W I 53706, and *Biochemistry Department, University of Wisconsin, Madison, W I 53706, U . S .A.
w O R D S . Low-voltage SEM, LTSEM, cryo-fixation, high-pressure freezing, cryo-coating, frozen-hydrated, freeze-drying, yeast, sea urchin embryos.
KEY
SUMMARY
Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM). Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize crossfractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (VO).In this study, the lowest possible Vo was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.52 kV. Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged. These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from highresolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice. Imaging of partially dehydrated (partially freeze-dried) samples, e.g. high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen-hydrated specimens. INTRODUCTION
Rapid freezing permits the fixation of biological samples under defined physiological
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conditions. It is generally acknowledged as the best initial fixation step because cryoimmobilization is much faster than immobilization with chemical fixation and therefore the sample remains closer to its living state (reviewed by Sitte et al., 1987; Moor, 1987). Imaging a cryo-fixed specimen in the frozen and fully hydrated state by use of a cold stage in the SEM is the most direct approach (Echlin, 1971). This method prevents artefacts related to chemical fixation and dehydration, such as shrinkage and collapse (reviewed by Read & Jeffree, 1991). It is therefore the method of choice for morphometric investigations. For high-resolution studies, more attention must be paid to reducing cryo-preparation artefacts that can be tolerated in studies at lower magnifications: in addition to the possibility of ice crystal formation during freezing, problems include water vapour contamination on the cold sample, artefacts related to coating the sample with a metal layer, as well as charging and mass loss of the hydrated specimen caused by the electron beam. Intramembranous particles have been visualized on frozen-hydrated cells (Walther et al., 1990)and tissue pieces (Herter et al., 1991) by using a dedicated cryo-sputtering system and a cold stage in a field-emission SEM (Muller et al., 1991). Similar results can be obtained with evaporation-coated frozen-hydrated samples (Wergin & Erbe, 1991). The highest resolution images of biological structures have been achieved with an inlens type field-emission SEM and thin transparent specimens, e.g. the 3-nm subunits of capsomeres of T-even phages (Hermann & Muller, 1991). Thus far, such a high resolution has not been achieved with whole cells and tissues because of the (poorly understood) interactions of the primary beam with the bulk of the sample. Our study further investigates the potential and limits of LTSEM of bulk biological samples by use of an in-lens field-emission SEM. The following problems were of special interest to us: what factors limit the resolution when imaging uncoated frozenhydrated samples? What are the current resolution limits when imaging coated frozenhydrated samples? What are the advantages and disadvantages of imaging partially dehydrated samples? MATERIALS A N D METHODS
Plunge freezing of yeast A pellet of commercially available bakers’ yeast cells (Saccharomyces cerevisiae) in the stationary growth phase was mounted on gold planchettes (Balzers Union, Balzers, Liechtenstein) and frozen by plunging into ethane cooled by liquid nitrogen. The frozen pellet was fractured under liquid nitrogen with a razor blade and the planchettes were mounted on a Gatan cryo-stage (Pleasanton, Calif.). In order to prevent water vapour or moisture contamination on the fracture face, the sample was kept in liquid nitrogen and the shutter of the cryo-stage was kept closed whenever possible. Some samples were directly transferred into a Hitachi S-900 SEM without coating (Figs. 1 and 3c); others were coated as described below. High-pressure freezing of Paramecium and sea urchin embryos High-pressure freezing (Moor & Riehle, 1968) allows for the cryo-fixation of relatively large biological samples (diameter about 1 mm; thickness up to about 0.4 mm) with minimal or no freezing artefacts (Moor, 1987). No chemical pre-fixation or cryopreservation is necessary, and therefore the sample can be frozen under defined physiological conditions. A commercial high-pressure freezer (HPM 010; Balzers Union) was used. Samples were frozen according to the protocol of Studer et al. (1989) using hexadecene to optimize pressure transfer. A droplet of the suspension containing either sea urchin embryos (24 h after fertilization) or P . tetraurelia was pipetted into the 3-mm gold or aluminium planchettes (Bal-Tec). A second planchette was put on the top and all cavities were filled with hexadecene. Hexadecene is not miscible with water and
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therefore does not act as a cryo-protectant (Studer et al., 1989). It may cause allergic reactions (Tobler & Freiburghaus, 1991), and therefore skin contact was avoided. Cryo-coating and cryo-transfer Some samples were coated in an MED 010 planar magnetron sputtering device (Balzers Union), equipped with a cold finger and an adaptor to accept cold specimens mounted on a Gatan cryo-transfer-stage, as developed by Hermann & Miiller (1991; H R 010, Balzers Union). This device is evacuated with a turbo molecular pump backed by a rotary pump so that the cold specimens are coated at a very low partial water vapour pressure. The fractured sample, mounted on the Gatan stage, was transferred into the cryocoating unit and cooled. The fracture face was protected from the moisture in the air by the shutter and by a small amount of evaporating nitrogen trapped between the shutter and the specimen. The transfer from liquid nitrogen into the specimen preparation chamber took about 5 s before the pumps could be started. After about 10 min, a Pa was reached, pure argon was introduced and sputter coating vacuum of 1 x began using a platinum target. The sputtering conditions were: argon gas pressure, 2 Pa; discharge current, 25 mA. After constant sputtering conditions had been established, the shutter was opened and the specimen was coated. The fully hydrated samples (Fig. 2) were coated immediately after opening the shutter at 148 K for 15 s. The Paramecium and sea urchin embryo samples (Fig. 4) were partially dehydrated ('etched') by opening the shutter at about 173 K for 10 min. Then the shutter was closed to start sputtering and, as soon as constant sputtering conditions were reached, the shutter was opened again and the sample was coated for 30 s. After coating, the shutter was closed again. The bell jar was flooded with argon gas and the Gatan cryo-stage with the sample was removed and transferred in cold nitrogen gas into the SEM. We made no direct measurement of the coat thickness, but, by comparison with data published previously (Walther et al., 1990), it is estimated to be below 3 nm for the fully hydrated samples (Fig. 2) and below 5 nm for the partially dehydrated samples (Fig. 4).
LTSEM A Hitachi S-900, in-lens, field-emission SEM (Nagatani et al., 1987) with modifications to enhance the performance of the microscope at low VO (Pawley & Erlandsen, 1989; Pawley, 1990) was used. T o minimize hydrocarbon residues in the vacuum, all rough pumping was performed by oil-free molecular drag pumps (Danielson, 1987). The microscope is equipped with a highly sensitive annular YAGdetector for BSE detection (Autrata, 1989). The Gatan stage was inserted into the SEM and the specimen chamber was pumped for about 10 min to remove the water vapour that entered the specimen chamber during transfer, then the shutter above the specimen was opened. Specimens were investigated at 138 K. The beam current was 1-5 x lo-" A. Electron doses were calculated according to the procedure of Echlin (1991). The Voand imaging modes required to obtain the individual figures are given in the figure legends. SE images were recorded at a scanning speed of 40 s, BSE images at a scanning speed of 80 s. RESULTS
Uncoated frozen samples The most direct approach is to look at fully hydrated and uncoated samples, thereby avoiding both drying and coating artefacts. Figure l(a) shows a low-voltage SE image of the protoplasmatic fracture face of a frozen-hydrated, uncoated yeast plasma membrane (primary magnification x 50,000). Vo (1 kV) was optimized in order to minimize charging artefacts. Intramembranous particles (IMPS) are not visible. A similar
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specimen was coated with platinum and imaged under the same conditions (Fig. la, inset), in order to demonstrate that the microscope at this low VOis capable of resolving these particles. On this coated sample the hexagonal pattern of the IMPs is clearly visible. Figure l(b) shows a similar uncoated sample imaged with the low-voltage BSE signal. The BSE image is less affected by local specimen charge-up and, at low voltage, the image provides reasonable contrast for a purely biological material. The characteristic invaginations are easily seen and the arrow points to a region where some even smaller structures appear on the fracture face. However, since the hexagonal pattern of the IMPs is not clearly resolved, it represents the actual resolution limit of the method. Figure l(c) shows an overview of the same fractured uncoated yeast sample (BSE image; Vo= 2-6 kV). The contrast is good enough to visualize the different types of fracture faces: protoplasmatic fracture faces (PF), extraplasmatic fracture faces (EF) and cross-fractured (CF) cells may be recognized. On a bulk specimen, the resolution with BSE is mainly limited by the excitation volume of the BSE signal. As this volume decreases with VO5, we hoped to obtain a better surface resolution by working at even lower Vo(Fig. Id; Vo= 2 kV). Unfortunately, the signal became very noisy, because the sensitivity of the BSE detector drops markedly below 2.5 kV (see Discussion). Partially dried, uncoated samples imaged with low-voltage BSE are represented in Fig. l(e) and (f). Figure l(e) shows a cross-fractured yeast cell. The nuclear envelope with nuclear pores can be recognized. The sea urchin embryo sample (Fig. l f ) shows a cross-fracture through the nucleus. The two membranes of the nuclear envelope can be clearly discerned.
Coated frozen-hydrated samples; imaging of IMPs The contrast and the visibility of structures was significantly enhanced by coating the sample with a thin layer of platinum. Figure 2(a) shows a protoplasmatic fracture face (SE image; Vo = 5 kV). The hexagonally arranged IMPs are visible (periodicity: 16.5 nm, according to Gross et al., 1978).The particles have small central holes (as described by Gross et al., 1978, using high-resolution T E M replicas). At this high magnification small cracks often appear in the platinum layer (electron dose of about 5000 e- nm-2). We also tried to image this sample with BSE (Fig. 2b). The best results were obtained at VO= 2.6 kV. However, contrast from the hexagonal particles is poorer than with the optimized SE image. Figure 2(c) and (d) represent examples of beam damage. Figure 2(c) is a first scan picture and Fig. 2(d) a similar area after irradiation for about 40 s corresponding to an electron dose of about 2500 e- nm-2 (one slow-speed scan for a picture). The membrane has been severely damaged by the first exposure to the electron beam.
Fig. 1. Uncoated samples. Fracture faces of yeast cells in the frozen-hydrated state not coated with a conductive layer. T h e SE image of a plasmatic fracture face of the plasmalemma (a) shows charging artefacts even at the low VOof 1 kV. Intramembranous particles are not visible. Inset: a similar sample at the same magnification but coated with platinum also imaged at VO= 1 kV. Now the hexagonal pattern of the IMPS is imaged. This indicates that the lack of information in the SE images of uncoated samples is a problem of contrast formation and is not caused by limitations in the performance of the SEM at low VO.(b) A similar area imaged with the low-voltage BSE signal ( V 0 = 2 . 6 kV). Charging effects are less visible. Beside the invaginations some surface particles also appear (arrow). However, resolution is limited due to the large escape depth of the BSE in the biological material at VO= 2.6 kV. (c) An overview of the same uncoated sample under the same conditions as (b). Plasmatic fracture faces (PF) and extraplasmatic fracture faces (EF) as well as cross-fractured cells (CF) are visible. (d) We hoped to improve resolution by further reducing VOto 2 kV, but the image became too noisy. (e) A partially dried, uncoated yeast cell with nuclear pores (arrow; BSE image, VO= 2.6 kV). (f) A partially dried uncoated sea urchin embryo cell (arrow depicts nuclear envelope; BSE image, VO= 2.6 kV).
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Fig. 2. Frozen-hydrated and platinum-coated bulk samples. Imaging of IMPSon the plasmatic fracture face. (a) High-magnification SE-image of the hexagonally arranged IMPS. T h e particles have a ring-like shape with a central depression. Small cracks appear in the platinum coat ( VO= 5 kV, electron dose about 5000 enm- ’). (b) BSE image; fewer details are visible than on the SE image ( VO= 2.6 kV). (c) T h e plasmatic fracture face at a primary magnification of x 70,000 (SE image). No cracks in the platinum layer are visible. (d) A similar area at the same magnification after having been exposed to the electron beam for one micrograph (electron dose about 2500 e - nm-’). T h e surface structure is destroyed by mass loss of the ice.
Drying artefacts and water vapour contamination Figure 3(a) and (b) show a specimen that was warmed to a temperature of 173 K for about 15 min, so that some of the surface water would sublime, as can be seen between the cells. At low magnifications, the plasmatic fracture face (PF) still looks well preserved (Fig. 3a). At higher magnifications, however (Fig. 3b), severe shrinking artefacts become visible. Water vapour is a major source of artefacts as has already been known from freeze
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Fig.3. Drying artefacts and water vapour contamination. T h e sample in (a) and (b) was warmed to about 173 K for 10 min. No drying artefacts are visible on the plasmatic fracture face at low magnification (a). At higher magnification (b), however, severe shrinkage effects are obvious (compare with Fig. 2c). (c) Ice crystals on top of fractured yeast cells formed by condensation of moisture, probably deposited as the specimen was being transferred from the liquid nitrogen to the SEM (uncoated samples imaged with the BSE signal). (d) Small particle contamination (white dots) caused by too high a partial water vapour pressure in the SEM. Arrows indicate the right edge of an area that was scanned by the electron probe before taking the picture.
fracture replica studies. Figure 3(c) shows a group of large ice crystals with either hexagonal or spherical shape. These contaminants were often observed on samples. We believe that they are originally formed by moisture trapped in the liquid nitrogen and cling to the specimen surface during specimen transfer.
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Another kind of water contamination is demonstrated in Fig. 3(d). The dots with a diameter of about 50 nm represent water vapour contamination that occurred in the SEM. The beam first irradiated the rectangular, inner area of the sample. The contaminants are removed within this area but the structure of the underlying membrane suffered from mass loss of water due to irradiation.
Partially dried samples Figure 4(a) and (b) show surface and fracture face of a Paramecium cell. Figure 4(c) and (d) show a portion of a sea urchin embryo cell. Figure 4(c) displays an area with a cross-fractured nucleus. The arrow marks the extraplasmatic fracture face of the outer membrane. Figure 4(d) shows a higher magnification view of the structures in the cytoplasm, with a vesicle surrounded by particles. Beam damage of partially dried samples was far less than with fully hydrated specimens. DISCUSSION
LTSEM of bulk frozen-hydrated samples is a complex problem and is influenced by various parameters. Water vapour contamination, coating, beam damage and drying affect the appearance of the sample at high magnifications. We found it useful to use bakers’ yeast, a well-known test specimen, in order to differentiate biological structures from preparation-related artefacts. The hexagonal pattern of IMPS in the plasmalemma of starved yeast can easily be discerned from preparation artefacts, e.g. water vapour deposition. Particles of condensed water vapour are randomly distributed on the specimen fracture faces in contrast to the hexagonal pattern. Also, shrinkage artefacts are easily recognized because the smooth appearance of well-preserved membranes with invaginations and hexagonal particles has been well documented in T E M replica studies (Moor & Miihlethaler, 1963; Gross et al., 1978).
Uncoated samples On uncoated samples, better and more predictable results were obtained with the low-voltage BSE signal than with the SE signal. Images at low and medium magnifications show good contrast. Resolution is surprisingly good, considering that the electrons are scattered only by the biological material and the ice. However, resolution is still poorer than with coated samples. We assume that resolution is limited by the escape depth of BSE. The escape depth of BSE is proportional to Vo513, and at 0.5 kV is about eight times smaller than at 2.6 kV (extrapolated from data of Joy, 1991). BSE operation at 0.5 kV should lead to significantly better resolution and would probably make the imaging of uncoated samples with very low-voltage BSE a competitive method. Unfortunately, the sensitivity of the BSE detector used in the present study drops below 2.6 kV, as seen in Fig. l(d), so we were unable to verify this approach. However, other laboratories are working on equipment that will allow for high-resolution BSE detection at 0.5 kV (Postek et al., 1990). Coated samples At high magnifications (Fig. 2a) the hexagonally arranged IMPS show a ring-like shape with a central hole. This confirms earlier high-resolution T E M replica studies (Gross et al., 1978). On hydrated and coated samples, higher spatial resolution was obtained using the SE signal rather than the BSE signal. This seems to conflict with our earlier studies (Walther & Hentschel, 1989; Walther et al., 1990), where we recommended the use of the high-resolution BSE signal for dried and double-layer coated samples. The explanation is the large mass difference between a dried and a hydrated sample. On a dried sample less scattering events occur in the bulk part of the
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Fig. 4. High-pressure frozen and partially dried samples. (a, b) Paramecium cell. (a) Low magnification of a fractured cell. CF = cross-fractured region; SF = surface of the ciliated cell. (b) Higher magnification showing mitochondria; arrow indicates a double membrane. (c, d) Portion of a cell in a sea urchin embryo. (c) A region of the nuclear envelope (arrow shows an extraplasmatic fracture face of the outer membrane). (d) High magnification of the same sample showing an area in the cytoplasm with vesicles surrounded by particles (arrow). Image mode for Fig. 4: SE; V, = 2 kV.
sample, and therefore the contrast is mainly formed by the overlying metal coat (Walther et al., 1990). However, on a hydrated sample, as used in this study, backscattering from the ice obscures the backscattered signal from the metal coat (Fig. 2b).
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In this study we used platinum as a coating material, because it had already been used in earlier studies (Walther et al., 1990; Muller et al., 1991), because the thin films have sufficient electrical conductivity to prevent charging, and because it sputters at relatively low energies, thereby reducing the danger of heat damage to the sample (Robards et al., 1981). We could not observe heat damage caused by the sputtering process. We therefore assume that it is not a limiting factor of this method. Finer grain sizes than are obtained with platinum have been reported using chromium (Peters, 1986; Hermann & Muller, 1991), tungsten (Lindroth & Sundgren, 1989) or by electron gun evaporation of platinum-iridium-carbon (Wepf et al., 1991). Whether these coating materials also improve the resolution possible on bulk frozen samples needs to be investigated. As noted earlier, a major problem of imaging fully hydrated samples at high magnification is the mass loss caused by electron irradiation (Walther et al., 1990). This mass loss, however, is dramatically reduced with partially dehydrated samples (Fig. 4).
Water vapour contamination Large contaminating ice crystals, e.g. visible in Fig. 3(c), are surprisingly stable. The large hexagonal or spherical ice crystals did not change their shape over a period of more than 30 min when the specimen was warmed to approximately 183 K during observation, whereas the underlying biological material showed clear evidence of sublimation (partial freeze-drying). T h e presence of overlying ice contamination, therefore, does not provide any information about the hydration state of the sample. Poor thermal contact between the sample and the overlying ice crystals is one explanation for this phenomenon. The ice crystals may preserve a lower temperature than the sample during warming. A more speculative explanation would attribute the phenomenon to differences in the vapour pressure associated with differences in the crystalline form between the ice in the biological sample and that in the overlying crystals (Livsey et al., 1991). Partially dehydrated samples Observation of partially dehydrated samples may become the main application of high-resolution LTSEM in biology. Its advantages over the other cryo-preparation techniques are: more intracellular structures are visible than with fully hydrated samples; beam damage is far less than with fully hydrated samples; in contrast to conventional fixation methods, the technique presented here does not involve any chemical treatment and therefore considerably reduces this source of preparation artefacts; and in contrast to the TEM replica technique, no difficult replica cleaning is needed. The main disadvantage of partially dehydrated samples compared to fully hydrated ones is the possibility of shrinkage artefacts. As demonstrated by comparing Fig. 3(b) with Fig. 2(b), shrinkage can occur at temperatures as low as 173 K. A membrane fracture face, such as the PF of the yeast plasmalemma, consists of only a lipid monolayer. Such a structure seems to be extremely unstable and disintegrates as soon as parts of the supporting ice are removed (Fig. 3b). Other structures, such as crossfractured bilayers and proteins attached to membranes, are probably more stable (Fig. 4). At present we do not know the extent to which the pattern visible in the cytoplasm (Fig. 4d) represents the in vivo situation or is the result of freezing or dehydration effects. Another limitation is that, as only water sublimes, other soluble components such as salts may obscure the surface after drying. This problem can be overcome with the freeze-substitution, critical-point drying approach (Barlow & Sleigh, 1979; Walther & Hentschel, 1989).
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ACKNOWLEDGMENTS
Sea urchin embryos were provided by Professor Gerald P. Schatten, Director of the Integrated Microscopy Resource (IMR). We thank all staff members of the IMR for generous support. Paul Walther was partially supported by a grant of the Swiss National Science Foundation. This work was also supported by NIH Grant DRR-570 to the Integrated Microscopy Resource, Madison, Wis.
REFERENCES
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High-resolution scanning electron microscopy of frozen-hydrated cells by P A U LWALTHER, YA CHEN,L O U I S L. PECH* and J A M E S B. P A W L E Y , Integrated Microscopy Resource, 1675 Observatory Dr., Madison, W I 53706, and *Biochemistry Department, University of Wisconsin, Madison, W I 53706, U . S .A.
w O R D S . Low-voltage SEM, LTSEM, cryo-fixation, high-pressure freezing, cryo-coating, frozen-hydrated, freeze-drying, yeast, sea urchin embryos.
KEY
SUMMARY
Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM). Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize crossfractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (VO).In this study, the lowest possible Vo was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.52 kV. Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged. These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from highresolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice. Imaging of partially dehydrated (partially freeze-dried) samples, e.g. high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated; and finally, the mass loss due to electron beam irradiation is greatly reduced compared to fully frozen-hydrated specimens. INTRODUCTION
Rapid freezing permits the fixation of biological samples under defined physiological
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conditions. It is generally acknowledged as the best initial fixation step because cryoimmobilization is much faster than immobilization with chemical fixation and therefore the sample remains closer to its living state (reviewed by Sitte et al., 1987; Moor, 1987). Imaging a cryo-fixed specimen in the frozen and fully hydrated state by use of a cold stage in the SEM is the most direct approach (Echlin, 1971). This method prevents artefacts related to chemical fixation and dehydration, such as shrinkage and collapse (reviewed by Read & Jeffree, 1991). It is therefore the method of choice for morphometric investigations. For high-resolution studies, more attention must be paid to reducing cryo-preparation artefacts that can be tolerated in studies at lower magnifications: in addition to the possibility of ice crystal formation during freezing, problems include water vapour contamination on the cold sample, artefacts related to coating the sample with a metal layer, as well as charging and mass loss of the hydrated specimen caused by the electron beam. Intramembranous particles have been visualized on frozen-hydrated cells (Walther et al., 1990)and tissue pieces (Herter et al., 1991) by using a dedicated cryo-sputtering system and a cold stage in a field-emission SEM (Muller et al., 1991). Similar results can be obtained with evaporation-coated frozen-hydrated samples (Wergin & Erbe, 1991). The highest resolution images of biological structures have been achieved with an inlens type field-emission SEM and thin transparent specimens, e.g. the 3-nm subunits of capsomeres of T-even phages (Hermann & Muller, 1991). Thus far, such a high resolution has not been achieved with whole cells and tissues because of the (poorly understood) interactions of the primary beam with the bulk of the sample. Our study further investigates the potential and limits of LTSEM of bulk biological samples by use of an in-lens field-emission SEM. The following problems were of special interest to us: what factors limit the resolution when imaging uncoated frozenhydrated samples? What are the current resolution limits when imaging coated frozenhydrated samples? What are the advantages and disadvantages of imaging partially dehydrated samples? MATERIALS A N D METHODS
Plunge freezing of yeast A pellet of commercially available bakers’ yeast cells (Saccharomyces cerevisiae) in the stationary growth phase was mounted on gold planchettes (Balzers Union, Balzers, Liechtenstein) and frozen by plunging into ethane cooled by liquid nitrogen. The frozen pellet was fractured under liquid nitrogen with a razor blade and the planchettes were mounted on a Gatan cryo-stage (Pleasanton, Calif.). In order to prevent water vapour or moisture contamination on the fracture face, the sample was kept in liquid nitrogen and the shutter of the cryo-stage was kept closed whenever possible. Some samples were directly transferred into a Hitachi S-900 SEM without coating (Figs. 1 and 3c); others were coated as described below. High-pressure freezing of Paramecium and sea urchin embryos High-pressure freezing (Moor & Riehle, 1968) allows for the cryo-fixation of relatively large biological samples (diameter about 1 mm; thickness up to about 0.4 mm) with minimal or no freezing artefacts (Moor, 1987). No chemical pre-fixation or cryopreservation is necessary, and therefore the sample can be frozen under defined physiological conditions. A commercial high-pressure freezer (HPM 010; Balzers Union) was used. Samples were frozen according to the protocol of Studer et al. (1989) using hexadecene to optimize pressure transfer. A droplet of the suspension containing either sea urchin embryos (24 h after fertilization) or P . tetraurelia was pipetted into the 3-mm gold or aluminium planchettes (Bal-Tec). A second planchette was put on the top and all cavities were filled with hexadecene. Hexadecene is not miscible with water and
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therefore does not act as a cryo-protectant (Studer et al., 1989). It may cause allergic reactions (Tobler & Freiburghaus, 1991), and therefore skin contact was avoided. Cryo-coating and cryo-transfer Some samples were coated in an MED 010 planar magnetron sputtering device (Balzers Union), equipped with a cold finger and an adaptor to accept cold specimens mounted on a Gatan cryo-transfer-stage, as developed by Hermann & Miiller (1991; H R 010, Balzers Union). This device is evacuated with a turbo molecular pump backed by a rotary pump so that the cold specimens are coated at a very low partial water vapour pressure. The fractured sample, mounted on the Gatan stage, was transferred into the cryocoating unit and cooled. The fracture face was protected from the moisture in the air by the shutter and by a small amount of evaporating nitrogen trapped between the shutter and the specimen. The transfer from liquid nitrogen into the specimen preparation chamber took about 5 s before the pumps could be started. After about 10 min, a Pa was reached, pure argon was introduced and sputter coating vacuum of 1 x began using a platinum target. The sputtering conditions were: argon gas pressure, 2 Pa; discharge current, 25 mA. After constant sputtering conditions had been established, the shutter was opened and the specimen was coated. The fully hydrated samples (Fig. 2) were coated immediately after opening the shutter at 148 K for 15 s. The Paramecium and sea urchin embryo samples (Fig. 4) were partially dehydrated ('etched') by opening the shutter at about 173 K for 10 min. Then the shutter was closed to start sputtering and, as soon as constant sputtering conditions were reached, the shutter was opened again and the sample was coated for 30 s. After coating, the shutter was closed again. The bell jar was flooded with argon gas and the Gatan cryo-stage with the sample was removed and transferred in cold nitrogen gas into the SEM. We made no direct measurement of the coat thickness, but, by comparison with data published previously (Walther et al., 1990), it is estimated to be below 3 nm for the fully hydrated samples (Fig. 2) and below 5 nm for the partially dehydrated samples (Fig. 4).
LTSEM A Hitachi S-900, in-lens, field-emission SEM (Nagatani et al., 1987) with modifications to enhance the performance of the microscope at low VO (Pawley & Erlandsen, 1989; Pawley, 1990) was used. T o minimize hydrocarbon residues in the vacuum, all rough pumping was performed by oil-free molecular drag pumps (Danielson, 1987). The microscope is equipped with a highly sensitive annular YAGdetector for BSE detection (Autrata, 1989). The Gatan stage was inserted into the SEM and the specimen chamber was pumped for about 10 min to remove the water vapour that entered the specimen chamber during transfer, then the shutter above the specimen was opened. Specimens were investigated at 138 K. The beam current was 1-5 x lo-" A. Electron doses were calculated according to the procedure of Echlin (1991). The Voand imaging modes required to obtain the individual figures are given in the figure legends. SE images were recorded at a scanning speed of 40 s, BSE images at a scanning speed of 80 s. RESULTS
Uncoated frozen samples The most direct approach is to look at fully hydrated and uncoated samples, thereby avoiding both drying and coating artefacts. Figure l(a) shows a low-voltage SE image of the protoplasmatic fracture face of a frozen-hydrated, uncoated yeast plasma membrane (primary magnification x 50,000). Vo (1 kV) was optimized in order to minimize charging artefacts. Intramembranous particles (IMPS) are not visible. A similar
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specimen was coated with platinum and imaged under the same conditions (Fig. la, inset), in order to demonstrate that the microscope at this low VOis capable of resolving these particles. On this coated sample the hexagonal pattern of the IMPs is clearly visible. Figure l(b) shows a similar uncoated sample imaged with the low-voltage BSE signal. The BSE image is less affected by local specimen charge-up and, at low voltage, the image provides reasonable contrast for a purely biological material. The characteristic invaginations are easily seen and the arrow points to a region where some even smaller structures appear on the fracture face. However, since the hexagonal pattern of the IMPs is not clearly resolved, it represents the actual resolution limit of the method. Figure l(c) shows an overview of the same fractured uncoated yeast sample (BSE image; Vo= 2-6 kV). The contrast is good enough to visualize the different types of fracture faces: protoplasmatic fracture faces (PF), extraplasmatic fracture faces (EF) and cross-fractured (CF) cells may be recognized. On a bulk specimen, the resolution with BSE is mainly limited by the excitation volume of the BSE signal. As this volume decreases with VO5, we hoped to obtain a better surface resolution by working at even lower Vo(Fig. Id; Vo= 2 kV). Unfortunately, the signal became very noisy, because the sensitivity of the BSE detector drops markedly below 2.5 kV (see Discussion). Partially dried, uncoated samples imaged with low-voltage BSE are represented in Fig. l(e) and (f). Figure l(e) shows a cross-fractured yeast cell. The nuclear envelope with nuclear pores can be recognized. The sea urchin embryo sample (Fig. l f ) shows a cross-fracture through the nucleus. The two membranes of the nuclear envelope can be clearly discerned.
Coated frozen-hydrated samples; imaging of IMPs The contrast and the visibility of structures was significantly enhanced by coating the sample with a thin layer of platinum. Figure 2(a) shows a protoplasmatic fracture face (SE image; Vo = 5 kV). The hexagonally arranged IMPs are visible (periodicity: 16.5 nm, according to Gross et al., 1978).The particles have small central holes (as described by Gross et al., 1978, using high-resolution T E M replicas). At this high magnification small cracks often appear in the platinum layer (electron dose of about 5000 e- nm-2). We also tried to image this sample with BSE (Fig. 2b). The best results were obtained at VO= 2.6 kV. However, contrast from the hexagonal particles is poorer than with the optimized SE image. Figure 2(c) and (d) represent examples of beam damage. Figure 2(c) is a first scan picture and Fig. 2(d) a similar area after irradiation for about 40 s corresponding to an electron dose of about 2500 e- nm-2 (one slow-speed scan for a picture). The membrane has been severely damaged by the first exposure to the electron beam.
Fig. 1. Uncoated samples. Fracture faces of yeast cells in the frozen-hydrated state not coated with a conductive layer. T h e SE image of a plasmatic fracture face of the plasmalemma (a) shows charging artefacts even at the low VOof 1 kV. Intramembranous particles are not visible. Inset: a similar sample at the same magnification but coated with platinum also imaged at VO= 1 kV. Now the hexagonal pattern of the IMPS is imaged. This indicates that the lack of information in the SE images of uncoated samples is a problem of contrast formation and is not caused by limitations in the performance of the SEM at low VO.(b) A similar area imaged with the low-voltage BSE signal ( V 0 = 2 . 6 kV). Charging effects are less visible. Beside the invaginations some surface particles also appear (arrow). However, resolution is limited due to the large escape depth of the BSE in the biological material at VO= 2.6 kV. (c) An overview of the same uncoated sample under the same conditions as (b). Plasmatic fracture faces (PF) and extraplasmatic fracture faces (EF) as well as cross-fractured cells (CF) are visible. (d) We hoped to improve resolution by further reducing VOto 2 kV, but the image became too noisy. (e) A partially dried, uncoated yeast cell with nuclear pores (arrow; BSE image, VO= 2.6 kV). (f) A partially dried uncoated sea urchin embryo cell (arrow depicts nuclear envelope; BSE image, VO= 2.6 kV).
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Fig. 2. Frozen-hydrated and platinum-coated bulk samples. Imaging of IMPSon the plasmatic fracture face. (a) High-magnification SE-image of the hexagonally arranged IMPS. T h e particles have a ring-like shape with a central depression. Small cracks appear in the platinum coat ( VO= 5 kV, electron dose about 5000 enm- ’). (b) BSE image; fewer details are visible than on the SE image ( VO= 2.6 kV). (c) T h e plasmatic fracture face at a primary magnification of x 70,000 (SE image). No cracks in the platinum layer are visible. (d) A similar area at the same magnification after having been exposed to the electron beam for one micrograph (electron dose about 2500 e - nm-’). T h e surface structure is destroyed by mass loss of the ice.
Drying artefacts and water vapour contamination Figure 3(a) and (b) show a specimen that was warmed to a temperature of 173 K for about 15 min, so that some of the surface water would sublime, as can be seen between the cells. At low magnifications, the plasmatic fracture face (PF) still looks well preserved (Fig. 3a). At higher magnifications, however (Fig. 3b), severe shrinking artefacts become visible. Water vapour is a major source of artefacts as has already been known from freeze
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Fig.3. Drying artefacts and water vapour contamination. T h e sample in (a) and (b) was warmed to about 173 K for 10 min. No drying artefacts are visible on the plasmatic fracture face at low magnification (a). At higher magnification (b), however, severe shrinkage effects are obvious (compare with Fig. 2c). (c) Ice crystals on top of fractured yeast cells formed by condensation of moisture, probably deposited as the specimen was being transferred from the liquid nitrogen to the SEM (uncoated samples imaged with the BSE signal). (d) Small particle contamination (white dots) caused by too high a partial water vapour pressure in the SEM. Arrows indicate the right edge of an area that was scanned by the electron probe before taking the picture.
fracture replica studies. Figure 3(c) shows a group of large ice crystals with either hexagonal or spherical shape. These contaminants were often observed on samples. We believe that they are originally formed by moisture trapped in the liquid nitrogen and cling to the specimen surface during specimen transfer.
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Another kind of water contamination is demonstrated in Fig. 3(d). The dots with a diameter of about 50 nm represent water vapour contamination that occurred in the SEM. The beam first irradiated the rectangular, inner area of the sample. The contaminants are removed within this area but the structure of the underlying membrane suffered from mass loss of water due to irradiation.
Partially dried samples Figure 4(a) and (b) show surface and fracture face of a Paramecium cell. Figure 4(c) and (d) show a portion of a sea urchin embryo cell. Figure 4(c) displays an area with a cross-fractured nucleus. The arrow marks the extraplasmatic fracture face of the outer membrane. Figure 4(d) shows a higher magnification view of the structures in the cytoplasm, with a vesicle surrounded by particles. Beam damage of partially dried samples was far less than with fully hydrated specimens. DISCUSSION
LTSEM of bulk frozen-hydrated samples is a complex problem and is influenced by various parameters. Water vapour contamination, coating, beam damage and drying affect the appearance of the sample at high magnifications. We found it useful to use bakers’ yeast, a well-known test specimen, in order to differentiate biological structures from preparation-related artefacts. The hexagonal pattern of IMPS in the plasmalemma of starved yeast can easily be discerned from preparation artefacts, e.g. water vapour deposition. Particles of condensed water vapour are randomly distributed on the specimen fracture faces in contrast to the hexagonal pattern. Also, shrinkage artefacts are easily recognized because the smooth appearance of well-preserved membranes with invaginations and hexagonal particles has been well documented in T E M replica studies (Moor & Miihlethaler, 1963; Gross et al., 1978).
Uncoated samples On uncoated samples, better and more predictable results were obtained with the low-voltage BSE signal than with the SE signal. Images at low and medium magnifications show good contrast. Resolution is surprisingly good, considering that the electrons are scattered only by the biological material and the ice. However, resolution is still poorer than with coated samples. We assume that resolution is limited by the escape depth of BSE. The escape depth of BSE is proportional to Vo513, and at 0.5 kV is about eight times smaller than at 2.6 kV (extrapolated from data of Joy, 1991). BSE operation at 0.5 kV should lead to significantly better resolution and would probably make the imaging of uncoated samples with very low-voltage BSE a competitive method. Unfortunately, the sensitivity of the BSE detector used in the present study drops below 2.6 kV, as seen in Fig. l(d), so we were unable to verify this approach. However, other laboratories are working on equipment that will allow for high-resolution BSE detection at 0.5 kV (Postek et al., 1990). Coated samples At high magnifications (Fig. 2a) the hexagonally arranged IMPS show a ring-like shape with a central hole. This confirms earlier high-resolution T E M replica studies (Gross et al., 1978). On hydrated and coated samples, higher spatial resolution was obtained using the SE signal rather than the BSE signal. This seems to conflict with our earlier studies (Walther & Hentschel, 1989; Walther et al., 1990), where we recommended the use of the high-resolution BSE signal for dried and double-layer coated samples. The explanation is the large mass difference between a dried and a hydrated sample. On a dried sample less scattering events occur in the bulk part of the
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Fig. 4. High-pressure frozen and partially dried samples. (a, b) Paramecium cell. (a) Low magnification of a fractured cell. CF = cross-fractured region; SF = surface of the ciliated cell. (b) Higher magnification showing mitochondria; arrow indicates a double membrane. (c, d) Portion of a cell in a sea urchin embryo. (c) A region of the nuclear envelope (arrow shows an extraplasmatic fracture face of the outer membrane). (d) High magnification of the same sample showing an area in the cytoplasm with vesicles surrounded by particles (arrow). Image mode for Fig. 4: SE; V, = 2 kV.
sample, and therefore the contrast is mainly formed by the overlying metal coat (Walther et al., 1990). However, on a hydrated sample, as used in this study, backscattering from the ice obscures the backscattered signal from the metal coat (Fig. 2b).
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In this study we used platinum as a coating material, because it had already been used in earlier studies (Walther et al., 1990; Muller et al., 1991), because the thin films have sufficient electrical conductivity to prevent charging, and because it sputters at relatively low energies, thereby reducing the danger of heat damage to the sample (Robards et al., 1981). We could not observe heat damage caused by the sputtering process. We therefore assume that it is not a limiting factor of this method. Finer grain sizes than are obtained with platinum have been reported using chromium (Peters, 1986; Hermann & Muller, 1991), tungsten (Lindroth & Sundgren, 1989) or by electron gun evaporation of platinum-iridium-carbon (Wepf et al., 1991). Whether these coating materials also improve the resolution possible on bulk frozen samples needs to be investigated. As noted earlier, a major problem of imaging fully hydrated samples at high magnification is the mass loss caused by electron irradiation (Walther et al., 1990). This mass loss, however, is dramatically reduced with partially dehydrated samples (Fig. 4).
Water vapour contamination Large contaminating ice crystals, e.g. visible in Fig. 3(c), are surprisingly stable. The large hexagonal or spherical ice crystals did not change their shape over a period of more than 30 min when the specimen was warmed to approximately 183 K during observation, whereas the underlying biological material showed clear evidence of sublimation (partial freeze-drying). T h e presence of overlying ice contamination, therefore, does not provide any information about the hydration state of the sample. Poor thermal contact between the sample and the overlying ice crystals is one explanation for this phenomenon. The ice crystals may preserve a lower temperature than the sample during warming. A more speculative explanation would attribute the phenomenon to differences in the vapour pressure associated with differences in the crystalline form between the ice in the biological sample and that in the overlying crystals (Livsey et al., 1991). Partially dehydrated samples Observation of partially dehydrated samples may become the main application of high-resolution LTSEM in biology. Its advantages over the other cryo-preparation techniques are: more intracellular structures are visible than with fully hydrated samples; beam damage is far less than with fully hydrated samples; in contrast to conventional fixation methods, the technique presented here does not involve any chemical treatment and therefore considerably reduces this source of preparation artefacts; and in contrast to the TEM replica technique, no difficult replica cleaning is needed. The main disadvantage of partially dehydrated samples compared to fully hydrated ones is the possibility of shrinkage artefacts. As demonstrated by comparing Fig. 3(b) with Fig. 2(b), shrinkage can occur at temperatures as low as 173 K. A membrane fracture face, such as the PF of the yeast plasmalemma, consists of only a lipid monolayer. Such a structure seems to be extremely unstable and disintegrates as soon as parts of the supporting ice are removed (Fig. 3b). Other structures, such as crossfractured bilayers and proteins attached to membranes, are probably more stable (Fig. 4). At present we do not know the extent to which the pattern visible in the cytoplasm (Fig. 4d) represents the in vivo situation or is the result of freezing or dehydration effects. Another limitation is that, as only water sublimes, other soluble components such as salts may obscure the surface after drying. This problem can be overcome with the freeze-substitution, critical-point drying approach (Barlow & Sleigh, 1979; Walther & Hentschel, 1989).
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ACKNOWLEDGMENTS
Sea urchin embryos were provided by Professor Gerald P. Schatten, Director of the Integrated Microscopy Resource (IMR). We thank all staff members of the IMR for generous support. Paul Walther was partially supported by a grant of the Swiss National Science Foundation. This work was also supported by NIH Grant DRR-570 to the Integrated Microscopy Resource, Madison, Wis.
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