Journal of Microscopy, Vol. 163, P t 1,July 1991,p p . 43-50. Received 8 M a y 1990;revised and accepted 4 September 1990

Application of high-resolution scanning electron microscopy to biological macromolecules

by TAKASHI NAKADERA AKIRA , M I T S U S H I Mand A K E I I C H IT A N A K A , Department of Anatomy, Tottori University School of Medicine, Yonago city, Tottori Prefecture, 683 Japan KEY w O R D S . Biological macromolecules, SEM, metal impregnation, haemocyanin, ferritin, thyroglobulin, immunoglobulin M.

SUMMARY

The development of ultrahigh-resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy-metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyroglobulin and immunoglobulin M were successfully imaged. Ultrahigh-resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules. INTRODUCTION

The structure of biological macromolecules has been studied by X-ray diffraction, and by negative staining, shadowing and freeze-replica methods with the transmission electron microscope. The scanning electron microscope (SEM) has not been used, despite the advantage of providing vivid, seemingly three-dimensional images, because of a combination of insufficient instrument resolution and lack of adequate methods for specimen preparation. In 1985 an ultrahigh-resolution SEM (UHS-T1) was developed (Tanaka et al., 1985, 1986), which was equipped with a field emission source and an objective lens of very short focal length. It had a resolution of 0 5 n m , theoretically adequate to observe macromolecules. Indeed some macromolecules including ribosomes, ferritin, and immunoglobulins have been imaged (Tanaka et al., 1989). In addition, intramembrane particles (Osumi et al., 1988), immunogold particles (Yasuda et al., 1989) and Fab fragments of IgG (Peters, 1989) have been observed using SEMs of the same type (Hitachi S-900, JEOL JSM-890). However, routine preparation methods for observation of macromolecules have not yet been established. We

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T . Nakadera, A . Mitsushima and K . Tanaka

previously reported a method of observing macromolecules without metal coating (carbon plate method, Tanaka et al., 1989) in order to avoid 'snowing' or decoration artefacts which are extremely difficult to avoid at this resolution. This method was effective for some molecules, but was not adequate for others which showed less contrast and were more easily damaged by the electron beam. Here we describe methods for dealing with these problems, and demonstrate their applicability to a number of biological macromolecules. MATERIALS A N D METHODS

Materials Haemocyanin (Buccinum; Polysciences, Inc., U.S.A.) was diluted with 0.07 M phosphate-buffered saline (PBS) with 10 mM sodium acetate and 25 mM MgC1, to a protein content of 1.8 mg/ml. Ferritin (horse spleen; Polysciences, Inc.) was diluted with a solution of 0 . 1 5 NaCl ~ and 25mM MgCl, to a protein content of 100-300pg/ml. Apoferritin (horse spleen; Sigma Chemical Co., U.S.A.) was diluted with 0.15 M NaCl and 25 mM MgCl, to a protein content of 25 pg/ml. Thyroglobulin (Bovine; a gift from D r K. Hirai) was diluted with 0.02 M ammonium acetate (pH 8.5) to a protein content of 1 mg/ml. Immunoglobulin M, prepared from normal human serum (ICN Immuno Biologicals, U.S.A.), was diluted with 0 . 1 5 ~NaCl and 25mM MgCl,, or 0 . 0 2 ~ ammonium acetate with 25 mM MgCl, (pH 8.5) to a protein content of 50pg/ml. Methods Droplets of the materials were placed on carbon-coated carbon plates (CC plates; Tanaka et al., 1991) and left for 1-3min. The CC plates were made by slicing commercial carbon rods (3 and 5 mm in diameter; Nisshin E M Co., Ltd, Japan) and polished with fine polishing films. After boiling in distilled water for about 10 min to remove oil, the CC plates were vacuum evaporated with carbon on their surface and were made hydrophilic by irradiation overnight with UV light. The specimens were briefly rinsed in distilled water and then impregnated with one of the following solutions of heavy-metal salts: O.l-l.OO,, uranyl acetate (UA) for 1 min, 1-2O,, phosphotungstic acid (PTA) for 1 min or 0.2",, tannic acid and 0.5-1.0°, osmium tetroxide (TO) for l m i n each. The specimens were briefly rinsed in distilled water and the excess fluid was blotted off with filter paper. They were then plunged into liquid ethane for rapid freezing. The frozen specimens were transferred into liquid nitrogen and dried in a freeze dryer with a turbomolecular pump (EIKO Engineering Co. Ltd, Japan). The dried specimens were observed with an ultrahigh-resolution SEM (UHS-T1) without metal coating at an accelerating voltage of 15kV. The electron probe current was 1.1 x lo-'' A and the exposure was 100 s. The total radiation doses were 6.4 x lo3 e/nm2at 100,000 x and 1.6 x lo5 e/nm2at 500,000 x . A heating stage was often used in order to reduce contamination, i.e. the specimens on the plates were heated at 333 K for 2 h in the specimen exchange chamber. After cooling, the specimens were inserted in the specimen chamber and observed. RESULTS

Haemocyanin (Buccinum) The haemocyanin appeared as hollow cylinders, circular in cross-section with a quadrilateral longitudinal profile (Fig. 1A). Their diameter and height were about 27 and 29 nm respectively. Higher magnification views of the circular face showed a collar and a central hole whose diameter was about 4.5nm (Fig. lC), but central caps (Mellema & Klug, 1972) were not found. The collar contained circularly arranged blobs (Fig. lC, arrow). According to previous reports (Mellema & Klug, 1972; van Bruggen et al., 1981) the blobs display a fivefold rotational symmetry. In our

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Fig. 1. Buccinum haemocyanin. (A) Low-magnification view. Magnification = 160,000. The molecules appear as circles or quadrilaterals.(B) Magnified view of quadrilateral aspect. Several parallel arranged faint grooves are recognized, running obliquely to the long axis (arrow). (C) Magnified view of circular aspect. Collar and central hole can be seen. The collar contains circularly arranged blobs (arrow).Magnification = 800,000.

observation there seemed to be ten blobs, as reported by Fernandez-Moran et al. (1966). On the longitudinal profiles, several parallel grooves were recognized, which ran either obliquely (Fig. lB, arrow) or at a 90" to the long axis. These fine structures, such as the blobs on the collar and the grooves, were clearer in specimens treated with UA than in those impregnated with PTA or TO.

Ferritin (horse spleen) Ferritin appeared as roughly spherical bodies about lOnm in diameter (Fig. 2A). Though readily observed even in untreated specimens, detailed structures were clearer in specimens impregnated with heavy-metal salts, in particular with UA. Two, three or four subunits were then observed (Fig. 2B-E). Collin et al. (1988), using rotary shadowed ferritin from a homopteran insect, reported ferritin molecules showing three axes of symmetry (twofold, threefold, fourfold). Negative staining also produced molecules apparently divided into four subunits (Farrant, 1954). Our findings are consistent with these results. The apparent size of ferritin varied remarkably depending on the method of impregnation: untreated specimens showed a diameter of 9.5 & 0.5 nm, those impregnated with PTA 10.1 & 0.6nm, with UA 10.0 k 0 5 n m , with T O 12.8 k 0.8nm.

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Fig. 2. Horse spleen ferritin. (A) Low-magnification view. The molecules appear as roughly spherical bodies. Magnification = 320,000. (B) Magnified ferritin molecule which is divided into four subunits. (C) Molecules divided into three subunits. (D) Ferritin divided into two subunits. (E) Ferritin not divided into subunits. Magnification = 800,000.

Apoferritin (horse spleen) These molecules were roughly spherical, with a relatively smooth surface. They were about 10 nm in size in specimens impregnated with PTA (Fig. 3), increasing to 12.8nm after T O treatment. Apoferritins were difficult to observe as untreated specimens, because they showed low contrast and were easily damaged by the electron beam. After metal impregnation they have a high contrast and become beam-resistant. In specimens treated with UA, grooves were sometimes observed on the apoferritin surface, although not as distinctly as those of ferritin. Immunoglobulin M (human I g M ) It is known from negative staining and freeze-replica work that IgM consists of a central plate surrounded by five radially arranged Y-shaped units, as seen in immunoglobulin G (Feinstein & Munn, 1969; Parkhouse, 1970; Shelton & McIntire, 1970; Heuser, 1983). Although SEM observations of IgG have already been reported (Peters, 1989; Tanaka et al., 1989), IgM had not previously been imaged by SEM. Figure 4(A, B) shows IgM after impregnation with PTA or TO, showing a star-shaped form. Thyroglobulin (bovine) Descriptions of the shape of this molecule have varied, from a helix with two turns (Bloth & Bergquist, 1968) to a sphere or prolate ellipsoid (Labaw & Rall, 1968) or an

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Fig. 3. Horse spleen apoferritin, showing spherical bodies similar to ferritin but which are slightly smaller. Magnification = 300,000.

Fig. 4. Immunoglobulin M. T h e molecules show a star-like form constructed from a central plate and five radially arranged subunits. Magnifications = 500,000.

ovoid or cylindrical body (Berg, 1973; Berg & Dahlgren, 1974; Berg & Ekholm, 1975; Berg et al., 1980). I n our observations the molecule appeared as a roughly spindleshaped body about 25 nm in length and 13nm in width (Fig. 5A-D). I t consisted of a large subunit flanked by two smaller subunits (Fig. 5B-D). According to Berg (1973) the molecules were constructed from two three-cornered subunits. We often saw such units (Fig. 5E) but could not determine whether molecules were actually formed from these structures. Thyroglobulin molecules were also very readily damaged by the electron beam unless metal-impregnated. DISCUSSION

To observe biological macromolecules in the SEM, some important problems must be considered: first, how to obtain a sufficiently high instrumental resolution; secondly, how to obtain enough specimen contrast and conductivity without metal coating; and thirdly, how to avoid damage and contamination of specimens in the electron beam. Regarding the first point, the ultrahigh-resolution S E M (UHS-T1; Tanaka et al., 1985, 1986) has a resolution of 0.5 nm at 30 kV and 0.6 nm at 15 kV, and is therefore capable of observing large molecules if they are appropriately prepared. On the second question, the problem of conductivity, the usual solution of metal coating cannot be used because the metal particles, being generally larger than the resolution of the SEM,are seen as ‘pebbles’ which markedly distort the image of a macromolecule. I n

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Fig. 5. Bovine thyroglobulin molecules. (A) T h e molecules appear as roughly spindle-shaped bodies. Magnification = 400,000. ( R D ) High-magnification views, showing a large subunit flanked by two smaller subunits. (E) Triangular subunits. One thyroglobulin molecule may be constructed from two such subunits. Magnification = 600,000.

the present study we used a method with carbon-coated carbon plates (CC plates) as substrates instead of metal coating. The carbon emits markedly less backscattered electrons than silicon or aluminium, and hence the signal-to-noise ratio of the specimen images is much better on the CC plates than silicon plates or aluminium foil. We also tried to use carbon foil on standard electron microscope grids, but found that it was often broken during specimen preparation, and was heavily contaminated when observed at high magnifications. Therefore, we prefer the CC plates to another substrate for our observations. Spreading molecules on CC plates (Tanaka et al., 1989, 1991) allows some molecules to be observed without charging effects, but is not generally applicable. We have therefore taken the approach of using a solution as a source of impregnating or staining metal, to decrease charging and improve the electron yield. Phosphotungstic acid (PTA), uranyl acetate (UA), and osmium tetroxide mordanted with tannic acid (TO) were all found to be effective. T O was most effective in increasing contrast, followed by UA and then PTA, but TO, because of the effect of bound tannic acid, markedly increased the apparent size of the molecules. PTA had no measurable effect as compared to unstained specimens, and UA very little. All three impregnations were effective in reducing beam damage. UA was particularly effective for revealing the fine structure of haemocyanin (Fig. lB, C ) , but sometimes produced a contamination of small particles c. 2 nm in diameter.

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The third problem of specimen contamination under the electron beam is very serious, because the fine structural details are gradually obscured during observation at very high magnification. To reduce contamination we improved the vacuum of the Pa) and the specimen exchange specimen chamber (ultimate vacuum of 2 x Pa) by using turbomolecular pumps. Furthermore, a heated chamber (4 x specimen stage (at 333K) was used to reduce contamination originating from the specimens, by thoroughly degassing them before observation. By these measures the rate of contamination was much reduced, and clear pictures could be obtained. In addition, it was previously confirmed that the macromolecules we used did not deteriorate when heated under these conditions. We thus were able to observe several macromolecules in the SEM. We feel that ultrahigh-resolution scanning electron microscopy will become increasingly important in the study of the morphology of biological macromolecules. ACKNOWLEDGMENT

We would like to express our cordial thanks to Dr Sally Stowe (The Australian National University, Canberra) for her kind help with this manuscript. REFERENCES Berg, G. (1973)An electron microscopic study of the thyroglobulin molecule.3. Ultrastruct. Res. 42,324-336. Berg, G.,Bjorkman, U. & Ekholm, R. (1980) The structure of newly synthesized intracellular thyroglobulin

molecules. Mol. Cell. Endocrinol. 20, 87-98. Berg, G. & Dahlgren, K.E. (1974) T h e electron microscopical structure of thyroglobulin obtained by micropuncture of rat thyroid follicles. Biochim. Biophys. Acta, 359, 1-6. Berg, G. & Ekholm, R. (1975) Electron microscopy of low iodinated thyroglobulin molecules. Biochim. Biophys. Acta, 386, 422-431. Bloth, B. & Bergquist, R. (1968) The ultrastructure of human thryoglobulin. 3. Exp. Med. 128, 1129-1136. van Bruggen, E.F.J., Schutter, W.G., van Breemen, J.F.L., Bijlholt, M.M.C. & Wichertjes, T. (1981) Arthropodan and molluscan haemocyanins. Electron Microscopy ofProteins, Vol. 1 (ed. by J. R. Harris), pp. 1-38. Academic Press, London. Collin, O., Thomas, D., Flifla, M., Quintana, C. & Gouranton, J. (1988) Characterization of a ferritin isolated from the midgut epithelial cells of a homopteran insect, Philaenus spumarius L. Biol. Cell, 63, 297-305. Farrant, J.L. (1954) An electron microscopic study of ferritin. Biochim. Biophys. Acta, 13, 569-576. Feinstein, A. & Munn, E.A. (1969) Conformation of the free and antigen-bound IgM antibody molecules. Nature, 224, 1307-1309. Fernandez-Moran, H., van Bruggen, E.F.J. & Ohtsuki, M. (1966) Macromolecular organization of hemocyanins and apohemocyanins as revealed by electron microscopy. 3. Mol. Biol. 16, 191-207. Heuser, J.E. (1983) Procedure for freeze-drying molecules adsorbed to mica flakes. 3. Mol. Biol. 169, 155-195. Labaw, L.W. & Rall, J.E. (1968) The crystal packing of thyroglobulin. 3. Mol. Biol. 36, 25-29. Mellema, J.E. & Klug, A. (1972) Quaternary structure of gastropod haemocyanin. Nature, 239, 146-150. Osumi, M., Baba, M., Naito, N., Taki, A., Yamada, N. & Nagatani, T. (1988) High resolution, low voltage scanning electron microscopy of uncoated yeast cells fixed by the freeze-substitution method. 3. Electron Microsc. 37, 17-30. Parkhouse, R.M.E., Askonas, B.A. & Dourmashkin, R.R. (1970) Electron microscopic studies of mouse immunoglobulin M; structure and reconstitution following reduction. Immunology, 18, 575-584. Peters, K.-R. (1989) Ultra high resolution SEM at high voltage images individual Fab fragments applied as molecular label to cell surface receptors. Proc. 47th Ann. Meet. E M S A (ed. by G. W. Bailey), pp. 70-71. San Francisco Press, San Francisco. Shelton, E. & McIntire, K.R. (1970) Ultrastructure of the r M immunoglobulin molecule. 3. Mol. Biol. 47, 595-597. Tanaka, K., Matsui, I., Kuroda, K. & Mitsushima, A. (1985) A new ultra-high resolution scanning electron microscope (UHS-Tl). Biomedical SEM, 14, 23-25 (In Japanese). Tanaka, K., Mitsushima, A., Kashima, Y., Nakadera, T. & Osatake, H. (1989) Application of an ultrahighresolution scanning electron microscope (UHS-TI) to biological specimens. 3 . Electron Microsc. Technique, 12, 146-154. Tanaka, K., Mitsushima, A., Kashima, Y. & Osatake, H. (1986) A new high resolution scanning electron microscope and its application to biological materials. Proc. XIth Int. Cong. E M , Vol. 3 (ed. by T. Imura, S. Maruse and T. Suzuki), pp. 2097-2100. The Japanese Society of Electron Microscopists, Tokyo.

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Tanaka, K., Mitsushima, A., Yamagata, N., Kashima, Y. & Takayama, H. (1991) Direct visualization of colloidal gold-bound molecules and a cell-surface receptor by ultrahigh-resolution scanning electron microscopy. 3. Microsc. 161, 455-461. Yasuda, K., Aiso, S., Nagatani, T. & Yamada, M. (1989) Direct observation of immunoreactive sites and antibody molecules by ultrahigh-resolution scanning electron microscope. 3. Electron Microsc. Technique, 12, 155-159.

Application of high-resolution scanning electron microscopy to biological macromolecules.

The development of ultrahigh-resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequ...
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