High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method

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1 /. Electron Microsc, Vol. 37, No. 1, 17-30, 1988 High Resolution, Low Voltage Scanning Electron Microscopy of Uncoated Yeast Cells Fixed by the Freeze-Substitution Method Masako OSUMI,* Misuzu BABA,* Nobuko NAITO,* Akiko TAKI,* Naoko YAMADA** and Takashi NAGATANI*** 'Department of Biology, and ** Laboratory of Electron Microscope, Japan Women's University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo, 112 Japan '"Application Laboratory, Naka Works. Hitachi Ltd., 882, lchige, Katsuta, Ibaraki, 312 Japan (Received July 27, 1987; accepted October 22, 1987) Uncoated yeast cells prepared by the freeze-substltution fixation and cryofracturing method were studied witb an "In-lens" type field emission scanning electron microscope (FESEM) at low voltage (0.8-3 kv). The micrographs were compared with those obtained with a conventional FESEM and coated specimens, as well as witb transmission electron micrographs of ultrathin-sectioned yeast cells. The images of uncoated fractured cells taken at kv seem to show the highest fidelity and thus they presented their threedimensional structures more dynamically. The fine filaments produced on the outermost surface of the cell wall were also clearly observed witb a low-voltage scanning electron microscope. The filaments of a similar specimen taken at 20 kv after coating with Pt-C 2 nm in thickness, however, showed an enlarged and intertwined structure because of a masking and decoration effect due to coating. In addition to several intracellular organelles, free ribosomes in the cytoplasm and Intramembranous particles of the cell membrane were revealed by the "In-lens" FESEM and these images correspond well to those obtained with uitrathin sectioning and freeze-fracturing replicas in the transmission electron microscope. Key words = L\'SEM (low voltage scanning electron microscope): direct observation: uncoated specimen: freeze-substitution fixation: yeast cell INTRODUCTION phenomenon during SEM observations. r.... /CI-. n., Until now, an accelerating voltage of around In scanning electron microscopy (SEM), it.,.... r., i 25 kv has generally been used, especially to is well known that use of the proper metal -. c,..., observe small features in biological speccoating for non-conducting materials such.,.,,.,.,.,,.,.,.., lmens. 1 ' 1 ' This value is a compromise beas dried biological specimens gives better. tu c UJ-. jjru-i. tween the fine probe diameter needed for high contrast and higher resolution. In thus case, resolution obtained at high kv and high con. the topograph.c contrast originates mainly trast obtajned at low ky owjng tq reduced from a comb.nat.on of the local thickness penetration of the specimen by the electron variations of the coating on the specimen beam surface and from changes in the effective j n us j ng a fi e id emission scanning electron thickness due to topographic variations. The microscope (FESEM) having a probe diameter metal coating is, in general, an inevitable proc- of 2-3 nm at 30 kv from the early 1970's, we ess needed to avoid the electric charge-up often found some artifacts caused by the

18 M. OSUMI et al. metal coating, that is, masking, modifying, or decorating minute topographic details of the specimens. These artifacts have also been reported by other researchers.

2 18 M. OSUMI et al. metal coating, that is, masking, modifying, or decorating minute topographic details of the specimens. These artifacts have also been reported by other researchers.'* 1 We tried a variety of coating methods to achieve the thinnest and finest coating possible in order to minimize the granularity of the metal. One of the best methods we found was a coating with a 2 nm thick (mean thickness on the plane) platinum-carbon (Pt-C) film made by the electron beam evaporation method at high vacuum (~ 10" 8 Pa). 6 ' 01 It is evident, however, that even if we applied a thinner coating to the specimen surface, the resolution by means of SEM (in other words, the finest details we could reveal by the SEM) should not be better than that of the coating film itself. Moreover, a loss of the topographic contrast from the uppermost surface of the specimen due to the beam penetration at high accelerating voltage will remain a fundamental problem for high resolution SEM (HRSEM). To avoid this problem a low voltage SEM (LVSEM)has been proposed 7 " 81 from the early stages of SEM applications. The LVSEMs produced were successfully applied to observations in the semiconductor industry, such as photoresist patterns used in LSI processing. 10 " 111 However, the resolution obtained at low voltages (<5 kv) was not sufficient to attract much interest in the field of biology. Recently, an ultrahigh resolution SEM (UHRSEM) composed of a cold cathode field emission (FE) gun and a highly exited objective lens, the so-called "In-lens FESEM," has been developed. 1 ' 141 It has a fine probe, about 4 nm in diameter at 1 kv by calculation. With it we have been trying to develop techniques to use this "In-lens FESEM" as an LVSEM for direct observation of totally uncoated biological specimens. Recently, we succeeded in the freeze-substitution (FS) fixation of yeast cells. 141 Because the specimens obtained by FS fixation have good electrical conductivity, it has enabled both ultrathin sectioning and SEM observation. 18 " 181 In this study the LVSEM images obtained with an "In-lens FESEM" at kv were compared with those obtained with a conventional FESEM and coated specimens. Ultrathin-sectioned specimens were also observed with a transmission electron microscope (TEM). More detailed inner structures and more dynamic three-dimensional images than those obtained by the freeze-fracturing replica method 19 ' 201 for the yeast cells are presented, and the high fidelity of the specimen topography is discussed. MATERIALS AND METHODS Microorganisms and growth conditions. Cells of the hydrocarbon-utilizing yeast Candida tropicalis pk 233,*" the methanol-utilizing yeast C. boidinii,"' 1 their fusants (FUS-15), 281 and Saccharomyces cerevisiae w were used. The cells were propagated by a method previously described. 16iI1 ~" ) Electron microscopy. The cells were prepared by the FS fixation method described previously 151 ; the cells were placed on the surface of a copper disk (0.02 mm in thickness, 3 mm in diameter) or a copper EM grid (one hole grid), a copper EM grid (400 mesh) was put on it, and another copper disk or EM grid (one hole grid) was placed on the grid. The specimens sandwiched between two copper disks or grids were then plunged as rapidly as possible into liquid nitrogen-cooled Freon 23. They were then transferred to liquid nitrogen. Subsequently, the two copper grids were separated and soaked in freeze-substitution fluid, 4% osmium tetroxide in absolute acetone, at 80 C for 48 hr. After a gradual return to room temperature, the specimens were washed with absolute acetone three times. One of the grids was subjected to critical point drying with liquid carbon dioxide for an SEM specimen," and observed with a Hitachi conventional FESEM, S-800, and "In-lens FESEM," S-900, at kv, and at kv, respectively. To compare uncoated and coated images, coating was performed in a Balzers High

3 Q High Resolution, LVSEM of Uncoated Yeast Cells CPD T-rf Centrifugation Freeze-substitution SEM observati Embedding TEM observation Cryo-fracture Freon23 Fig. 1. Procedure of the freeze-substitution fixation method for SEM and TEM observations. CPD: critical point drying. (See in "Materials and Methods) Vacuum Freeze-Etch Unit BAF 301 with an Electron Beam Evaporator Equipment EVM 052 with an Electron Gun EK 552."" The other grid was conventionally passed through propylene oxide, embedded in a Quetol 812 mixture, ultrathin sectioned for a TEM specimen, 15 ' and observed with a Hitachi H-800 TEM at 200 kv. Thus one development in our FS fixation method is that specimens attached to one copper grid can be used for SEM and the ones on the other grid for TEM (Fig. 1). RESULTS AND DISCUSSION Observation with conventional FESEM The fine structure of uncoated yeast cells was found to be preserved in an extremely good condition within an accelerating voltage of kv when they were examined with the S-800 SEM* 4 '"' (Fig. 2). Although it depended on conductivity of the specimen at an accelerating voltage higher than 1.3 kv, the images showed the "charge-up" phenomenon which was already found in the image even at 1.0 kv (Fig. 2d). It was recognized that the outermost surface of the cell wall consists of fine filaments (Figs. 2, 3a and 4a). Figure 4b shows the image of a similar specimen at 20 kv after being coated with Pt-C of 2 run thickness. 28 ' Since this image reproduces the intertwined or coalesced condition of the filamentous structure at the outermost surface of the cell wall (arrow), it seems that the image of the uncoated specimen has more fidelity in revealing the cell ultrastructure (Fig. 3). The nucleus, mitochondria and microbodies are clearly seen in the cytoplasm of the fractured cell. In addition, unclear membrane pores were observed. These im-

4 20 M. OSUMT et al. ages correspond to those of intracellular organelles in the TEM images as shown in Fig. 3b. Shrinkage of the vacuole or exfoliation from the cell wall was occasionally observed (arrow). This appeared to derive from damage due to the critical point drying. Although the images formed at an accelerating voltage of kv seem to give the natural impression of the cell surface structure, a voltage between 1.0 and 1.1 kv can be considered to offer optimum image quality (Figs. 2 and 3a), since it gives better resolution. These C. tropicalis cells are gtown in n-alkane as the sole carbon source for 17 hr. A characteristic feature of n-alkane-growing yeast C. tropicalis is abundant formation of microbodies in its cytoplasm, together with a remarkable increase in catalase activity."' Microbodies multiply by division from preexisting microbodies, like mitochondria."' In our previous paper 27 ' and also in Fig. 3b it is hard to tell from the ultrathin-sectioned image in the TEM whether the microbodies are ball-like or sausage-type in appearance. However, in Fig. 3a, it seems that the concave and convex images of globules appearing in the cytoplasm are microbodies and they are ball-like in appearance. Figures 4b and 4c show a cell grown in n-alkane for 6 hr. There are usually fewer microbodies than in the cells grown for 17hr(Fig. 3b). 27) Observation with "In-lens" FESEM By using the UHRSEM, S-900, the cells show higher resolution and a higher-fidelity image"'"-"" (Figs. 5 and 6a). The double nuclear membrane and nuclear membrane pores were detected in the Candida cell grown in n-alkane. Part of the nucleus is thought to be a nucleolus judging from the different efficiency of the secondary electrons (Fig. 5). Figure 6 shows the image of the LVSEM of the fusant cell grown in methanol (a) and the corresponding TEM image (b), respectively. In Fig. 6a, the nucleus, six large and cubic microbodies, and a long mitochondrion are recognized. A characteristic microbody in methanol has a crystalloid in the matrix. The crystalloid is composed of a composite crystal consisting of two key enzymes of methanol oxidation, alcohol oxidase (10 nm in diameter) and catalase (7 nm in diameter), arranged alternately.' 1 " 841 The fractured microbodies (arrow) appeared in the inner structure of the crystalloid space, but were scarcely observed in the lattice structure which showed in the TEM image (Fig. 6b). However, the enzyme molecules (7 and 10 nm in diameter) are too small to be detected by secondary electrons even with the UHRSEM, for the deviation of the two sizes is only 1.5 nm. In addition, the very small particles (30 nm) appearing profusely in the cytoplasm seem to be ribosomes according to the corresponding TEM image (Fig. 6b). The images obtained at kv were not critically different from those appearing in the series examined with the S-800 SEM (Figs. 7a, 7b and 7d). Figure 7c is an image showing typically poor quality at more than 2-3 kv. Figures 7d and 7e are stereo images which gratifyingly reveal the threedimensional structure of the fusant cells grown in methanol. Figure 8 shows the fractured images of the cell surface of the fusant yeast cells grown in the medium containing methanol as the sole carbon source. 1 " The outermost cell surface appeared to consist of typical long filaments which showed a different image from C. tropicalis, one of the parent strains (Figs. 2 and 5) and other yeasts (data not shown). In the concave (probably PF face of the cell membrane by freeze replica) (Fig. 8a) and convex (probably EF face) (Fig. 8b) faces of the cell membrane, membrane imagination (indicated by CMI) can be observed. Furthermore, as a gratifying result, intramembranous particles (indicated by IMP) were detected. In the higher magnification images (Figs. 9a and 9b), there are many particles distributed homogeneously. The diameter of these intiamembranous particles is not uniform: the smallest

5 High Resolution, LVSEM of Uncoated Yeast Cells 21 one is about lonm and the largest about 20 nm. The particles present a more threedimensional image than is seen in freezereplica images, although the image cannot clarify the substructure of the particles. Figures 9c and 9d show the images of PF and EF faces of the cell membrane of C. tropicalis in which their intramembranous particles were slightly smaller than those of the fusant cell. This is the first identification of intramembranous particles by SEM. These images correspond to freeze-replica imagesi9,2o,!»). however, the number of these intramembranous particles in the PF face (Figs. 9a and 9c) appeared to be the same as those of the particles in the EF face (Figs. 9b and 9d), which usually has fewer than the PF face. 19 ' 36 ' The reason for this phenomenon should be determined by further studies. CONCLUSION High-fidelity SEM images were produced by applying low accelerating voltage to uncoated biological specimens fixed by the freeze-substitution method. In addition to several intracellular organelles, free ribosomes in the cytoplasm and intramembranous particles of the cell membrane were identified. The SEM image of fractured yeasts revealed a more undulated topography than the TEM images obtained by freeze-replica. Furthermore, the SEM image is especially helpful for interpretation of three-dimensional structures of intracellular organelles in appearance, such as microbodies whose biogenesis had not been revealed during the cell cycle. From the present results, their shapes in 6-hr and 17-hr cultures are preliminarily estimated to be globular. Further combination studies with LVSEM and TEM should reveal it more clearly. With this new information the previous model of the yeast cell"' was modified. 16 ' Optimum freezing is a critical problem in LVSEM for preparation of biological specimens. Future subjects for study include (1) development of a better method of preparing enhanced conductivity of biological specimens and (2) achievement of higher resolution at LV. Then we should be able to identify several organelles in the cell by SEM, such as ribosomes attached to the nuclear membrane, microtubules in the nucleus, the Golgi apparatus, and actin filaments, which had been revealed by the FS fixation method in the TEM. 16 ' In addition, (3) a conductive coating of high quality is also useful to increase the contrast in small features in biological specimens even in the LVSEM. Although several metal coatings have been studied for enhancing generation of surface information from small biological features less than a few nm in the HVSEM, 87 """ there is at present no useful way to determine a priori an appropriate thickness of the coating (or even the method itself) for a given surface complexity of biological specimens. Then, for an LVSEM, more careful studies for coating will be needed to give the best resolution. Acknowledgments. This work was supported by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation. REFERENCES 1) Osumi, M., Fukuzumi, F., Yamada, N., Nagatani, T., Teranishi, Y., Tanaka, A. and Fukui, S.: J. Ferment. Technol., 53, 244 (1974) 2) Osumi, M. and Torigata, S.: SEMI 1977 II, 617 (1977) 3) Peters, K.: SEM/1985 IV, 1519 (1985) 4) Rosowski, J. K., Roemer, S. C, Hoagland, K. D. and Roth, W. A.: SEM/1984 I, 29 (1984) 5) Osumi, M., Yamada, N., Nagano, M., Murakami, S., Baba, N., Oho, E. and Kanaya, K..: SEMI 1984 I, 11 (1984) 6) Yamada, N., Nagano, M., Murakami, S., Ikeuchi, M., Oho, E., Baba, N., Kanaya, K.. and Osumi, M.: J. Electron Microsc, 32, 321 (1983) 7) Pawley, J.: Proc. 42nd Ann. Meet. Electron Microsc. Soc. Am., 1984, p ) Joy, D.: Proc. 42nd Ann. Meet. Electron Microsc. Soc. Am., 1984, p ) Boyes, E. D.: Proc. 42nd Ann. Meet. Electron Microsc. Soc. Am., 1984, p ) Cotes, V. J.: Proc. 40th Ann. Meet. Electron

6 22 M. OSUMI et al. Microsc. Soc. Am., 1982, p ) Tuggle, D. W. and Watson, S. C: Proc. 42nd Ann. Meet. Electron Microsc. Soc. Am., 1984, p ) Watanabe, T. and Yamada, M.: J. Electron Microsc, 34, 215 (1985) 13) Nagatani, T. and Saito, S.: Xlth Int. Congr. Electron Microsc., Kyoto, 1986, p ) Nagatani, T., Saito, S., Yamada, M. and Sato, M.: Scan. Microsc. (SEM Int.), 1, 901(1987) 15) Baba, M. and Osumi, M.: J. Electron Microsc. Tech., 5, 249 (1987) 16) Baba, M. and Osumi, M.: J. Electron Microsc, 33, 303 (1984) 17) Baba, M. and Osumi, M.: J. Electron Microsc, 34, 222 (1985) 18) Osatake, H., Tanaka, K. and Inoue, T.: /. Electron Microsc. Tech., 2, 201 (1985) 19) Osumi, M.: in Yanagishima, N., Oshima, Y. and Osumi, M. (Eds.), Anatomy of Yeast Cells (in Japanese), Kodansha Scientific, Tokyo, 1981, p. 4 20) Nagano, M., Kodama, K., Baba, N. and Kanaya, K.: /. Electron Microsc, 31, 268 (1981) 21) Osumi, M., Miwa, N., Teranishi, Y., Tanaka, A. and Fukui, S.: Arch. Microbiol., 99, 181 (1974) 22) Fukui, S., Tanaka, A., Kawamoto, S., Yasuhara, S. and Osumi, M.: J. Bacteriol., 123, 317 (1975) 23) Kobori, H., Ohta, M., Kanakubo, M. and Osumi, M.: 60th Ann. Proc. Bacteriol., 1987, p ) Osumi, M., Baba, M., Suzuki, T., Watanabe, T. and Nagatani, T.: Bio-Med. SEM, 14, 47 (1985) EXPLANATION OF FIGURES 25) Watanabe, T., Nagatani, T., Yamada, M., Osumi, M. and Baba, M.: Bio-Med. SEM, 14, 1 (1985) 26) Osumi, M.: Proc. Xlth Int. Congr. Electron Microsc., 1986, p ) Osumi, M., Fukuzumi, F., Teranishi, Y., Tanaka, A. and Fukui, S.: Arch. Microbiol., 103, 1 (1975) 28) Osumi, M., lmaizumi, F., Imai, M., Sato, H. and Yamaguchi, H.: J. Gen. Appl. Microbiol., 21, 375 (1975) 29) Yamada, N., Nagatani, T. and Osumi, M.: Bio- Med. SEM, 15, 35 (1986) 30) Osumi, M. and Nagatani, T.: in Geiss, R. H. (Ed.), Microbeam-Anal. 1987, Hawaii, p ) Osumi, M. and Sato, M.: J. Electron Microsc, 27, 127 (1978) 32) Osumi, M., Sato, M., Sakai, T. and Suzuki, M.: J. Electron Microsc, 28, 295 (1979) 33) Osumi, M., Nagano, M., Yamada, N., Hosoi, J. and Yanagida, M.: J. Bacteriol., 151, 376 (1982) 34) Baba, N., Nagano, M., Osumi, M. and Kanaya, K.: J. Electron Microsc, 33, 203 (1984) 35) Yamaguchi, H., Iwata, K., Nagano, M. and Osumi, M.: J. Electron Microsc, 30, 305 (1981) 36) Osumi, M.: in Yanagishima, N., Oshima, Y. and Osumi, M. (Eds.), Anatomy of Yeast Cells, (in Japanese) Kodansha Scientific, Tokyo, 1981, p. 5 37) Tanaka, K.: Proc. Xlth Int. Congr. Electron Microsc., Kyoto, 1986, p ) Haggis, G. H.: Proc. Xlth Int. Congr. Electron Microsc., Kyoto, 1986, p ) Peters, K.-R.: SEM/1985, 1519 (1985) Fig. 2. LVSEM images of uncoated C. tropicalis grown in n-alkane at kv and examined with a high resolution SEM, S-800. x 6,400. Fig. 3. LVSEM image of uncoated C. tropicalis cell grown in n-alkane for 17 hr at 1.0 kv and examined with a S-800 (a) and corresponding TEM image (b). a, x21,000; b, x 16,800. Fig. 4. LVSEM (a), HVSEM (b), and corresponding TEM image (c) of C. tropicalis cells grown in n-alkane for 6 hr and examined with the S-800. a, A part of cell surface; b, HVSEM image of Pt-C of 2 nm coated C. tropicalis. a and b, x 28,000; c, x 19,800. Fig. 5. LVSEM image of uncoated C. tropicalis cell grown in n-alkane for 17 hr at 1.5 kv and examined with the UHRSEM, S-900. x 25,200. Fig. 6. LVSEM using S-900 (a) and corresponding TEM (b) images of fusant cells grown in methanol for 24 hr at 1.5 kv. a, x 27,000; b, X 27,600. Fig. 7. LVSEM images of uncoated fusant cells grown in methanol for 24 hr at 0.8 (a), 1.0 (b), 1.5 (d and e, stereo photograph, 0, 8 respectively), and 3 (c) kv and examined with the S-900. a-c, x 16,000; d and e, x 24,000.

High Resolution, LVSEM of Uncoated Yeast Cells 23 Fig. 8. LVSEM images of cell surface of fusant cells of the same specimen as in Fig. 7 at 1.5 kv using S-900. a, X42.400; b, X42.000. Fig. 9.

7 High Resolution, LVSEM of Uncoated Yeast Cells 23 Fig. 8. LVSEM images of cell surface of fusant cells of the same specimen as in Fig. 7 at 1.5 kv using S-900. a, X42.400; b, X Fig. 9. LVSEM images of intramembranous particles of cell membrane of fusant cells (a and b) and C. tropicalis cells (c and d). a and c, PF face; b and d, EF face, x 135,000. Abbreviations. CM, cell membrane; CMI, invagination of cell membrane; Cry, crystalloid; CW, cell wall; rer, rough endoplasmic reticulum; IMP, intramembranous particle; M, mitochondrion; Mb, microbody; MbM, microbody membrane; N, nucleus; NE, nuclear envelope; NP, nuclear pore; Nu, nucleolus; R, ribosome; V, vacuole; Ves, vesicle; VM, vacuolar membrane.

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Specimen Preparation Technique for a Microstructure Analysis Using the Focused Ion Beam Process

Specimen Preparation Technique for a Microstructure Analysis Using the Focused Ion Beam Process by Kozue Yabusaki * and Hirokazu Sasaki * In recent years the FIB technique has been widely used for specimen