Arch. Histol. Cytol., Vol. 55, Suppl. (1992) p. 5-15

Ultrahigh-Resolution of Biological

Keiichi Depatment Received

Summary.

TANAKA

The

Electron

Microscopy

Materials

and

of Anatomy, March

Scanning

Noboru

Faculty

YAMAGATA

of Medicine,

Tottori

University,

Yonago,

Japan

18, 1992

age

of

ultrahigh-resolution

In 1981, TANAKA suggested from his findings in biological materials that SEM resolution could surpass the theoretical limit if the beam spot of the SEM would be made smaller than the limit. KURODAet al. (1985) observed the crystal surface of a tungsten tip with a field emission TEM which had an SEM attachment, and showed a resolution below 1 nm. TANAKA et al. (1985, 1986) developed an Ultrahigh-resolution SEM (UHS-T1) together with Hitachi Ltd. It was equipped with a field emission gun and an objective lens of very short focal length, and achieved a resolution of 0.5 nm. With the development of this instrument, research fields amenable to SEM expanded from cellular structures to viruses and biological macromolecules. For example, AIDS viruses, TZ bacteriophages, ferritins, hemocyanins, and immunoglobulins were clearly observed (TANAKA et al., 1989; NAKADERA et al., 1991). On the other hand, with the improvement of instrumental resolution, some unexpected problems came to the fore in the area of specimen preparation, the most serious problem being that the conventional metal-coating method was no longer suitable, as metal particles coated on specimen surfaces were plainly seen as round "pebbles". To solve this problem, we devised a method called "the CC plate method" (TANAKA et al., 1991). With this method, specimens smaller than 10 nm in diameter could be observed without metal-coating. Besides metal-coatings, there are other problems to be overcome in observation at very high magnifications. These include contamination, beam damage and the contrast of specimens. In response, we took various countermeasures, including improvement of the vacuum in our SEM, and heavy metal impregnation of specimens. Biological macromolecules may now be as easily studied by SEM as are cellular fine structures.

scanning

electron microscopy (SEM) began in 1985, when the UHS-T1, with a resolution of 0.5 nm, was developed. Commercial instruments of the same or similar types followed rapidly. As instrumental resolution progressed, conventional specimen preparation methods became inadequate, and a number of new techniques were devised. In this paper, detailed procedures for these preparation methods such as the CC plate method and heavy metal impregnation are described, together with precautions recommended for achieving ultrahigh-resolution. Some applications of the method to biological specimens are also reported. Morphological identification of immunoglobulins prepared from human blood was attempted, and although the identification was not completely successful this technique may yet come to be of use in the clinical examination of allergic or infectious diseases. SEM images of complement, Clq, proteoglycan and the helical structure of double stranded DNA are shown, as also is the visualization of immunolabelled cell-surface receptors.

Over the last two decades, the scanning electron microscope (SEM) has come to be widely used in biomedical studies, since it provides seemingly three dimensional images which can be easily understood. Many studies on surface fine structures and intracellular structures of various kinds of cell and tissue have been carrid out. Until recently, however, the SEM was regarded as only a subsidiary instrument for ultrastructure research, because its resolution was markedly inferior to that of the transmission electron microscope (TEM), and it was generally accepted for years that, theoretically, the resolution of the SEM could not be improved under 1 nm because of the escape depth of secondary electrons from a solid surface. 5

6

K. TANAKA and N. YAMAGATA:

METHODS

2. Procedures

1) Samples of biological macromolecules were appropriately diluted with a suitable solution such as a buffer solution or a physiological saline solution. 2) Droplets of the samples were placed on CC plates (prepared as described below) and left for 3-5 min. 3) After adhering the macromolecules to the plates, excess fluid was removed with filter paper and the plate rinsed in distilled water for 1 min. 4) Either immediately or after heavy metal impregnation (to increase the resistance to beam damage and enhance the contrast), the specimens were dried by rapid freezing-freeze drying, critical point drying or t-butyl alcohol drying (INouE et al., 1989). For heavy metal impregnations (NAKADERA et al., 1991) the specimens were treated with uranyl acetate (0.1-2.0% solution, for 1 min) or osmium tetroxide mordanted with tannic acid (0.2% osmium, 0.2% tannic acid and then 0.2% osmium for 5 min each). The rapid freezing-freeze drying was carried out under the following conditions: The specimens were rapidly frozen in liquid ethane. Then they were transferred to liquid nitrogen in a small metal bucket, and the bucket was inserted in a freeze drier with a turbomolecular pump (EIKO Engineering Co. Ltd., Japan). The drying was performed for 45 mm at -85t, keeping a vacuum of 2 x 10-6 Torr (TANAKA et al., 1988). The other two drying methods were carried out by routine procedure.

CUTTING

Fig.

1.

3. Preparation

Schematic

5. CARBON

3.REMOVAL

drawing

COATING

of procedures

(Fig. 1).

methods

for various

samples

Immunoglobulins Ten ml of blood was collected intravenously from a healthy human. The blood was left standing in a test tube for 2 h and the isolated serum centrifuged at 3,000 rpm for 10 min. After the sediment was removed, the fluid was again centrifuged at 3,000 rpm for 10 min. The supernatant was collected and mixed with saturated ammonium sulfate until the concentration of ammonium sulfate was 33%. After the salting out of the globulin fraction, the solution was centrifuged at 10,000 rpm for 10 min. The sediment was collected and then dissolved in physiological saline solution. The fluid was again mixed with satu-

2. POLISHING

4. DRYING

the CC plates

These were made by slicing commercial carbon rods (5 mm or 3 mm in diameter, Nissin EM Co. Ltd, Japan) in 2 mm thick slices with a low speed bone saw (Isomet: Buehler Ltd., USA) (Fig. 1-1) and polished with fine polishing films (Imperial lapping film; 3M Co., USA) until the surface was a mirror face (Fig. l-2). After boiling in distilled water for about 10 min to remove fat (Fig. 1-3), the carbon plates were dried in an incubator (Fig. 1-4) and evaporated with carbon in a vacuum evaporator (Fig. 1-5). They were then made hydrophilic by irradiation overnight with UV light (Fig. 1-6). Such carbon plates are called carbon coated carbon plates, or, briefly, CC plates.

1. The CC plate method

1.

for making

6.

U.V.

for making

OF

FAT

IRRADIATION

the CC plate.

Ultrahigh-Resolution

rated ammonium sulfate to obtain a 33 % solution. The solution was again centrifuged at 10,000 rpm for 10 min and the sediment dissolved in physiological saline solution. This solution was used as material for micros4opy after it was diluted with physiological saline solution to a protein content of 500 ng/ml. In addition, the specimens were fixed with 1% glutaraldehyde in distilled water for 2 min after placing on the CC plate. Complement, Clq A commercial preparation of human Clq from Serva Feinbiochemica GmbH & Co. (Germany) was used as material after it was diluted with 1% ammonium acetate solution to a protein content of 10 U/ml. The specimens were treated with osmium tetroxide mordanted with tannic acid after they were placed onto the CC plate. Proteoglycan Purified bovine nasal cartilage proteoglycan aggregate and monomer were both commercial preparations purchased from ICN ImmunoBiologicals (USA). The samples were dissolved in sterilized phosphate buffer solution (a mixture of 0.02 M KH2PO4i 0.03 M K2H PO4, 0.1 M KCl and 10 mM EDTA : pH 7.0) by mixing in a stirrer for 8 h at 4t . The solution was diluted to a protein content 10 ng/ml and used as material. These specimens were stained with 0.2% uranium acetate solution for 1 min to enhance the image contrast. DNA The samples were prepared by a microspreading method (SEKI et al., 1984) slightly modified by us, and the CC plate method. Erythrocytes obtained from the blood of a 2-day-old chicken were washed with a CKM buffer (0.05 M Na-cacodylate, 0.025 M KCI, 0.005 M MgC12 and 0.25 M sucrose, pH 7.5) and suspended in a 0.2 M KCI solution (4 X 10' cells/ml). The suspension was treated with 10 volumes of 0.08% Joy detergent (Proctor and Gamble, Ohio, USA, pH 8.7) for 1 min. Then 1/10 volume of 10% formalin in 0.1 M sucrose was added. After standing for 30 min, the suspension was centrifuged in an Eppendorf tube containing the CC plate at 7,000 g for 5 min. After a brief rinsing in distilled water, the specimens were stained with 2 % aqueous uranyl acetate for 1 min. They were then dehydrated through a graded ethanol series and dried by critical point drying. All procedures before fixation with the formalin solution were performed at 0-4t.

SEM

7

Cell surface receptor A clonal macrophage cell line (A640-BB-2, TANIGAWAet al., 1983) was used. Cells were dispersed with trypsin and EDTA, cultured on the CC plates and incubated at 37t for 24 h. The macrophages were reacted with monoclonal antibodies for Mac-1 antigen (rat IgG2b, clone Ml/70; Hybritech Inc., USA) for 2 h at 4 t and protein A-colloidal gold (15 nm; E. Y. Laboratories, USA) for 4 h at 4t. The specimens were fixed in 1% glutaraldehyde for 10 min and treated with osmium tetroxide mordanted with tannic acid. Afterwards, cells were dehydrated through an ethanol series and critical point dried. Microscopy For observing biological macromolecules, it is desirable to use an ultrahigh-resolution SEM with a resolution under 1 nm. Incidentally, the main production design specifications of our SEM (UHS-T1) are as follows: Electron optics: Electron gun: field-emission electron source. Accelerating voltages : 1-30 kV Lens system: 2-stage electromagnetic lens system. Objective lens: f 3.6 mm; Cs 1.6 mm; Cc 2.0 mm. Specimen stage: Side-entry specimen stage (In-lens system) Resolution: 0.5 nm (Beam spot size on calculation: 0.45 nm) Magnification range: 150-1,000,000 x. Vacuum system : Gun chamber : ion pump (60 l/s) x 1. First and second intermediate chambers: ion pump (20 l/s) X 1 each. Specimen chamber: turbo-molecular pumps (340 l/s) x 1, (60 l/s) X 1 and rotary pump (174 1/mm) x 1(Connected in series). Specimen exchange chamber: turbo-molecular pump (60 l/s) X 1 and rotary pump x 1 in series. Ultimate vacuum: Electron gun chamber: 1 x 10-a Pa. First and second intermediate chambers: 3 x 108, 3 x 10-' Pa, respectively. Specimen chamber: 3 x 10-8 Pa. Specimen exchange chamber: 2 x 10-4 Pa. Anti-contamination devices: Cold finger x 1 and cold trap x 1. All specimens were mechanically attached to a specimen holder with a special apparatus (to avoid the use of silver paste). The obsevations were carried out without metal coating at accelerating voltages of 15-

8

K. TANAKA

and

N.

YAMAGATA:

2

A

B

D

C

E

F

3 Fig.

G 2.

Various

immunoglobulins

in human

blood

serum.

Fig. 3. Classified immunoglobulins which appeared in human blood serum. group of IgG, IgD and IgE, E: IgA (dimer), F, G and H: unknown bodies.

A: IgM, B, C and D: mixed

Ultrahigh-Resolution

SEM

9

30 kV, probe current of 1.3 x 10-11 A, electron irradiation dose of 3 x 10'-1.2 x 105 e/nm2.

RESULTS Immunoglobulins

(Igs)

Though it is well known that blood serum includes various kinds of immunoglobulin, among these IgG, IgA and IgM, the globulin fraction as a whole has not yet been used as a material for studying the morphology of Igs by electron microscopy, because it is difficult to identify each of the immunoglobulins only from their shapes. Therefore the initial stages of morphological research have until now usually employed chemically purified samples. We assume, however, that observation of the whole globulin fraction may have clinical significance in diagnosing or determing the prognosis of various diseases if we could simply analyze the morphology and quantitative relationship of the Igs in the serum of patients. In order to help establish the feasibility of such a technique, we made observations of Igs in the whole globulin fraction, analysed on the basis of the data reported so far from purified samples. In the specimens prepared from blood serum by salting-out, various kinds of immunoglobulin could be seen (Fig. 2), and these could in fact be divided into a number of reasonably distinct groups on the basis of their shapes (Fig. 3). The first group consisted of three subunits arranged in Y or V form (Fig. 3B). As the shapes were in agreement with those of IgG as reported in previous papers by SEM (TANAKA et al., 1989), negative staining (VALENTINE and GREEN, 1967: KENNETH and METZGER, 1982) and the freeze replica method (HEUSER, 1983), the central subunit was regarded as the Fc fragment and the two peripheral subunits as the Fab fragments. Their contour-sizes varied, however, considerably. The subunits of some appeared 1.5-2 times larger than the average size (Fig. 3C, D). Although the majority in this group belonged to IgG, it was not clear whether the larger ones were also IgG or not. On the basis of their shapes (BARKER et al., 1980; PUMPHREY, 1986), it might be predicted that IgD and IgE would also appear in this group, but there is presently no precise information available on their SEM images. To obtain such data, these globulins must probably first be indentified using an immunochemical method. There is a precedent for this, as YAMAGATA (1992) has successfully observed an IgG labelled with an immuno-gold complex. Application of the same tech-

Fig.

4.

Complement,

Clq.

sticks" radially arranged. has been slightly darkened

They

are found

as six

"drum

To focus attention, this photo except around two Clq.

nique to IgD and IgE may yield the degree of detailed information needed to perform a identification on a morphological basis. The second group showed an X-form which consisted of two IgG-like bodies connected at the central fragment (Fig. 3E); the contour size was fairly definite. Judging from shapes reported previously (PuMPHREY,1986), they were regarded as IgA (dimer). The third group consisted of particularly large shapes. They appeared to be constructed of five IgGlike bodies arranged radially from a central plate (Fig. 3A). In our previous study (NAKADERA et al., 1991), we observed chemically purified IgM samles by the same observation method. The shape of this group was consistent with the results of that study, and bodies of this type were therefore identified as IgM. In addition to these well known immunoglobulins, some other unknown bodies were found in our specimens. The first consisted of five spherical subunits whose sizes were smaller (3-4 nm in diameter) than those of the IgG subunit and the subunits arranged in hairpin-like form (Fig. 3F). The second group were constructed of two larger (7 nm in diameter) paired subunits (Fig. 3G) and the third showed an irregularly bent cord-like structure (Fig. 3H). Although these profiles had shapes which were sufficiently characteristic to enable them to be grouped, we have as yet no means of identifying them.

10

K. TANAKA and N. YAMAGATA:

B

A

C Fig.

5.

Proteoglycan

view

of a portion

C. Proteoglycan

molecules.

of a proteoglycan monomers.

Arrow

A. Proteoglycan

aggregate,

aggregate.

Mucopolysaccaride

shows

terminal

portions

We are further still unable to unequivocally identify all immunoglobulins in the blood serum from their morphology. However, even if such a "perfect" analysis is impossible, it is evident that a great deal of information can already be gained. We believe that in the near future the morphological analysis of immunoglobulins may be of great use in the clinical examination of allergic or infectious diseases, for example, immunodeficiency syndrome. Complement,

Clq

Clq is a glycoprotein which consists of a six-polypeptide chain. In a proposed model (KENNETH et al., 1976) based on TEM studies using negative stainings (SHELTON et al., 1972; KNOBEL et al., 1975), Clq was shown as a bunch of flowers. The six "flowers", or

showing

a centipede-like

chains

are visible

of mucopolysaccaride

structure. around

B. Magnified

the protein

cores.

chains.

polypeptide chains, branched radially from the root of the bunch (central portion), and each chain had a peripheral globular portion (5 nm x 7 nm) at its end. In our observations Clq molecules showed the same appearance. They were viewed as complexes constructed like six radially arranged drum sticks (Fig. 4). The measured lengths of their contours (32-35 nm) were almost the same as those calculated using the model (37 nm). Complement, Clq is one of the most difficult materials for us to observe by SEM, because the width of its polypeptide chains is remarkably fine (1.5 nm). Proteoglycan Proteoglycan, known that

is also a kind of glycoprotein; it is a proteoglycan aggregate molecule

Ultrahigh-Resolution

SEM

11

A

B

C Fig. 6. Chromatin fibers and DNA. A. Chromatin fibers spread out from a chicken erythrocyte nucleus. Fibers without bead-like structures (DNA fiber; DF) and fibers with beads (nucleosome fiber; NF) are observed. B. Highly magnified DNA fiber, showing "left handed" helical structure. C. Highly magnified view of a nucleosome fiber, consisting of nucleosomes and linker-DNA. The linker DNA shows a "right handed" appearance (From INAGA et al., 1991).

12

K. TANAKA

and

N.

YAMAGATA:

A

B Fig. 7. Cell surface receptor (macrophage C3bi) bound with an immunogold complex. A. Secondary electron imaging. Arrow shows the boundary of the two subunits in the receptor. B. The same field of view but with backscattered electron imaging. The bright spot corresponds to the gold in the immunogold complex. (From TANAKA et al., 1991).

shows a centipede-like structure (ROSENBERGet al., 1975; MORGELIN et al., 1988). In the molecules, filaments of hyaluronic acid make the backbones, and proteoglycan subunits of varying length arise laterally at fairly regular intervals from the opposite sides of the backbones. The proteoglycan subunits consist of a filamentous protein core, and mucopolysaccharide chains which attach laterally to this protein core. In the present study, we observed the proteoglycan backbone and the subunits, which had various lengths and projected at approximately right angles from the backbone (Fig. 5A), though the subunits generally showed not a filamentous but rather a rough cord-like structure because of the tangle of mucopolysaccharide chains on the core proteins. In some molecules, however, fine filamentous structures were observed (Fig. 5B). These filaments might correspond to extended mucopolysaccharide chains. On the other hand, proteoglycan monomers appeared as irregulary bent fibers (Fig. 5C). On the side of the fibers, some protrusions which arose from the fiber at fairly regular intervals were visible (Fig. 5C, arrow). These protrusions might correspond to the terminal portions of the mucopolysaccharide chains.

DNA Many attempts by various techniques with TEM have been made to visualize directly the DNA double helix, but this has been difficult or impossible by conventional SEM because of its poor resolution. With the recent advances in resolution, it has become feasible to observe DNA by SEM. In our specimens prepared from chicken erythrocyte nuclei (INAGA et al., 1991), two types of chromatin fibers were recognized: fine fibers bearing beadlike structures (nucleosomes), shown in Figure 6A, (NF), and other DNA fibers without beading (Fig. 6A, DF). At higher magnifications, the DNA fibers showed a "left-handed" double-stranded helical structure of 2.5-5.0 nm diameter and a rather irregular pith of 3.5 to 4.5 nm (Fig. 6B). Nucleosome fibers consisted of nucleosomes (appearing as spherical or ellipsoidal particles of irregular size) joined by 5-25 nm lengths of linker DNA, about 2.5-4.0 nm in diameter (Fig. 6 C). The twisted and "bumpy" appearance of linker DNA was judged to be the effect of a "right-handed" double helix with a pitch of 4.5 nrn. It was interesting that the "left handed" structure could be found in DNA fibers which had no associated nucleosomes. According to NICKOL et al. (1982), the

Ultrahigh-Resolution

transformation from B-DNA (right handed) to ZDNA (left-handed) is reversible in nucleosome formation, and only B-DNA can form the complete nucleosome structure, the association of nucleosomes and the normal chromatin assembly being disrupted when B-DNA transits to Z-DNA. Therefore it might be expected that no nucleosomes were observed on the "left handed" DNA in our study. Cell surface

receptor

In plasma membranes there are many kinds of cellsurface receptors which bind to specific molecules with high affinity. Cell-surface receptors can be observed in non-metal-coated specimens by SEM, when the specimens have been heavily stained with osmium tetroxide, because the receptors in this case produce relatively strong SE emission. Therefore, if a receptor is to be reacted with an already known antibody and a gold-colloid for labelling and then treated with osmium, the receptor bound to the immuno-gold complex should be visible, and it would be possible to recognize the property of the receptor. In a previous paper (TANAKA et al., 1991), we reacted a cultured macrophage with monoclonal antibodies for Mac 1 antigen (AULK and SPRINGER, 1980) and protein A-gold. Subsequently, the attachment of a gold-particle via the antibody to a round granule on the cell surface was seen (Fig. 7). As the Mac 1 antigen is identical to the murine complement C3bi receptor (BELLER et al., 1982; WRIGHT et aL, 1983), this granule can be characterized as a C3bi receptor. The receptor is a complex composed of two polypeptides, and the granule may be that part of the dimer exposed on the plasma membrane. A faint groove distinguishable on the granule (Fig. 7, arrow) might be the boundary of the two subunits. Although we could identify only one receptor in this study, we believe that it should be possible with this technique to identify many kinds of receptors or channels by using various antibodies.

DISCUSSION In order to observe very tiny specimens such as biological macromolecules by an ultrahigh-resolution SEM, there are both necessary precautions to be taken and some problems to be overcome. First one must find a way to give electrical conductivity to the specimens. Since dried specimens are particularly subject to charging, they should be provided with electrical conductivity by some method. For this, they have usually been coated with metals. However,

SEM

13

metal particles coated on specimen surfaces are plainly seen as rounded "pebbles" in high-resolution observation. Though it has been reported that chromium coating by a special technique gives a good result in avoiding this problem (MULLER and HERMANN, 1988; HERMANN et al., 1991), we devised the CC plate method and tried to observe specimens without any metal coating. In this method, almost all incident electrons penetrate through the specimens and are removed through the carbon plates; consequently, residual electrons in the specimens and emitted electrons from them are balanced, and no charging of the specimens takes place. In addition, carbon emits markedly less backscattered electrons than silicon or aluminium, and hence the signal-to-noise ratio of the specimen images on the CC plates is much better than that on silicon plates or aluminium foils. By this method, therefore, we could clearly observe some macromolecules such as ferritin and IgG without the metal-coating that is liable to obscure the true surfaces and ditails of the specimens (TANAKA et al., 1989). However, there are some biological macromolecules which can not be successfully observed merely by using the CC plate method, because they exhibit lower contrasts even on the carbon plates and are more susceptible to damage by an electron beam. The second problem, then, is how to enhance their contrast and increase their resistance to beam damage. For such macromolecules, we applied heavy metal impregnation methods using phosphotungustic acid, uranyl acetate or osmium tetroxide mordanted by tannic acid. These methods were effective, and apof erritin, thyroglobulins, immunoglobulin M (IgM) and others were observed for the first time by SEM (NAKADERAet al., 1991). The third problem is specimen contamination by the electron beam. The fine structure of tiny specimens is liable to be "snowed" or deformed very quickly at very high magnifications. It is then very important to reduce the contamination. Although the precise mechanism of the contamination remains unsetteled, it is thought that the contamination occurs because of polymerides which are produced by interaction between the electron beam and residual hydrocarbon molecules in the vacuum (YADA, 1981). Two approaches that can be used to reduce contamination depend on improving the vacuum around the specimen, and reducing the sources of contamination in the immediate environment. To obtain a clean and higher vacuum, the use of turbo-molecular pumps instead of diffusion pumps is recommended. We remarkably improved the vacuum of the specimen chamber and the specimen exchange chamber of our

14

K. TANAKA and N. YAMAGATA:

SEM by using turbo-molecular pumps (See main specifications of our SEM). The other of these approaches is reducing the contamination originating near the specimen. The use of a silver paste for attaching specimens to holders must be avoided, because the vapor arising from the paste is one of the most serious sources of contamination. Specimens should be fixed mechanically in the specimen holder (we use a special holder made for the CC plate). The use of a heating stage is also effective in decreasing contamination. In our study, some specimens were heated at 60t for 2 h on a heating stage in the specimen exchange chamber for degassing. After cooling, the specimens were inserted in the specimen chamber and observed. The technique used for taking photographs is also very important for reducing contamination. To minimize exposure to the beam, we usually took a new field, that is, we adjusted the astigmatism at an image of a neighboring area, moved the area, and photographed a new field of view after quick refocusing. Skill in SEM-operation is required for doing this technique rapidly and in correct sequence. When these measures are meticulously carried out, less contaminated, clearer pictures can be obtained in ultrahigh-resolution scanning electron microscopy.

in the biomedical

field.

Acknowledgement. We express our cordial thanks to Dr. Sally STOWE, Australian National University, Canberra, for her kind help with this manuscript.

REFERENCES AULK,

K. A.

and

T. A.

SPRINGER:

BARKER,

W. C., L. K.

KETCHAML and

REMARKS

BELLER,

D.

I.,

T. A.

SPRINGER

and

Anti-Mac-1 selectively inhibits type three complement receptor. 1009 (1982). HERMANN,

of a

0.

DAYHOFF:

chain

domains.

R.

SCHREIBER:

D.

J.

the mouse and human J. Exp. Med. 156: 1000-

R., H. SCHWARZ and M. MULLER:

High

preci-

sion immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold. J. Struct. Biol. 107: 38-47 (1991). HEUSER,

J.

absorbed (1983).

E.:

Procedure

to mica

for

flakes.

freeze-drying

J. Mol.

Biol.

K. TAKAKA:

molecules

169: 155-195

SEM

images

of

DNA double helix and nucleosomes observed by ultrahigh-resolution scanning electron microscopy. J. Electron Microsc. 40: 181-186 (1991). INOUE,

Observation methods with electron microscopes usually advance by the palallel progression of instrumental resolution and the development of specimen preparation. In recent years, SEM resolution has been startlingly improved by the development of ultrahighresolution SEM. In biological work, instrumental resolution is no longer the current limiting factor, and is sufficient to allow the viewing of large molecules. As a consequence, new problems have become evident in specimen preparation, and solutions to old problems have become inadequate. To address these problems in visualizing macromolecules rather than subcellular structures, the CC plate method and the heavy metal impregnation technique, others were developed. It is expected that the further improvement of SEM-resolution will happen in not only SE but also the backscattered electron mode and, following this, the devising of various more precise preparation methods, for example, a labelling technique with very small gold particles for chemically definite positions in specimens. If such advances could be achieved, we feel that ultrahigh-resolution SEM will become increasingly important in the study of ultrastructures

M.

Origins of immunoglobulin heavy Mol. Evol. 15: 113-127 (1980).

INAGA, S., H. OSATAKE and

CONCLUDING

Cross-reaction

rat-antimouse phagocyte-specific monoclonal antibody (Anti-Mac-1) with human monocytes and natural killer cells. J. Immunol. 126: 359-364 (1980).

T.,

H.

OSATAKE

freeze-drying ning electron 249 (1989). KENNETH,

H.

and

H.

TAKAHASHI:

A

new

instrument using t-butyl alcohol for scanmicroscopy. J. Electron Microsc. 38: 246R. and

D. W. METZGER:

Immunoelectron

microscopic localization of idiotypes and allotypes on immunoglobulin molecules. J. Immunol. 129: 2548-2553 (1982). KENNETH,

B., M. REID and R. R. PORTER:

Subunit

com-

position and structure of subcomponent Clq of the first component of human complement. Biochem. J.155:1923 (1976). KNOBEL,

H. R., W. VILLIGER

analysis and electron prepared by different 82 (1975). KURODA,

and H.

ISLIKER:

K., S. HOSOKI and T. KoMODA:

crystal surface of W (110) field emitter Electron Microsc. 34: 179-182 (1985). MORGELIN, M., M. PAULSSON, HEINEGARD and J. ENGEL: -Assembly with hyaluronate

ied by electron (1988).

Chemical

microscopy studies of human Clq methods. Eur. J. Immunol. 5 : 78-

microscopy.

Observation

of

tip by SEM. J.

T. E. HARDINGHAM, D. Cartilage proteoglycans and link protein as stud-

Biochem.

J. 253: 175-185

MULLER, M. and R. HERMANN: High fling electron microscopy of biological

resolution scanspecimens. In:

(ed. by) G. W. BAILEY: Proc.

46th Ann. Meet. EMSA,

Ultrahigh-Resolution

Milwaukee, San Francisco (p. 186-187).

Press,

San Francisco,

1988

and

NAKADERA, T., A. MITSUSHIMA

and K. TANAKA:

Applica-

tion of high-resolution scanning electron microscopy to biological macromolecules. J. Microsc. 163: 43-50 (1991). NICKOL, J.,

M.

BEHE and

G. FELSENFELD:

Effect

of the

B-Z transition in poly (dG-me5dC)poly (dG-me5dC) on nucleosome formation. Proc. Nat. Acad. Sci. USA 79: 1771-1775 (1982). PUMPHREY,

R.:

Computer

models

of the human

immuno-

flexibility.

Immunol.

globulins-Shape and segmental Today 7: 174-178 (1986). ROSENBERG,

L., W. HELLMANN and A. K. KLEINSCHMIDT:

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J. Biol. Chem. 250: 1877-1883 (1975). SEKI, S., T. NAKAMURA

and T. ODA: Supranucleosomal

fiber loops of chicken erythrocyte tron Microsc. 33: 178-181 (1984). SHELTON,

E.,

K. YONEMASU

and

chromatin.

R.

M.

J. Elec-

STROUD:

structure of the human complement component, Proc. Nat. Acad. Sci. USA 69: 65-68 (1972). TANAKA, by

high

K.:

Demonstration

resolution

of

scanning

intracellular electron

Ultra-

Clq.

structures

microscopy.

1981/II. SEM Inc., AMF O'Hare,

H.

OSATAKE:

USA, 1981 (p. 1-8).

K. KURODA and A. MITSUSHIMA:

A new ultrahigh resolution scanning electron microscope (UHS-T1) (In Japanese). Biomed. SEM 14: 23-25 (1985). TANAKA, K., A. MITSUSHIMA, Y. KASHIMA and H. OSATAKE:

15

Y. KASHIMA, T. NAKADERA

Application

of an

ultrahigh-resolu-

tion scanning electron microscope (UHS-T1) to biological specimens. J. Electron Microsc. Tech. 12: 146-154 (1989). TANAKA, K., A. MITSUSHIMA, and

H.

TAKAYAMA:

N. YAMAGATA, Y. KASHIMA

Direct

visualization

of

colloidal

gold-bound molecules and a cell-surface receptor ultrahigh-resolution scanning electron microscopy. Microsc. 161: 455-461 (1991).

by J.

TANIGAWA, T., H. TAKAYAMA, A. TAKAGI and G. KIMURA:

Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. 1. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA 640 mutant of simian virus 40. J. Cell Physiol. 116: 303-310 (1983). VALENTINE,

R.

C. and

N.

scopy of an antibody-hapten 615-617 (1967).

M.

GREEN:

complex.

Electron

micro-

J. Mol. Biol. 27:

WRIGHT, S. D., P. E. RAO, W. C. VAN VOORHIS, L. S. CRAIGMYLE, K. IIDA, M. A. TALLE, E. F. WESTAERG, G. GOLDSTEIN and S. C. SILBERSTEIN:

In:

(ed. by) Om JOHARI: Scanning electron microscopy/ TANAKA, K., I. MATSUI,

TANAKA, K., A. MITSUSHIMA,

SEM

C3bi using USA YADA, KEN

Identification

of the

receptor of human monocytes and macrophages by monoclonal antibodies. Proc. Nat. Acad. Sci. 80: 5699-5703 (1983). K.: Specimen contamination (In Japanese). DEN16: 2-10 (1981).

YAMAGATA,

N.:

Immunogold

colloid

particles

observed

by ultrahigh resolution scanning electron microscopy (In Japanese). J. Yonago Med. Ass. 43: 75-84 (1992).

A new high resolution scanning electron microscope and its application to biological materials. In: (ed. by) T. IMURA, S. MARUSE and T. SUZUKI: Proc. XIth Int. Congr. EM, Kyoto, Vol. 3. The Jap. Soc. E. M., Tokyo, 1986 (p. 2097-2100). TANAKA, K., A. MITSUSHIMA, UCHI: Development

of

Y. KASHIMA and H. YAMA-

a freeze

scanning electron microscopy SEM 17: 55-57 (1988).

drying

apparatus

(In Japanese).

for

Biomed.

Prof. Keiichi TANAKA Seirei Christopher College of Nursing 3453 Mikatagahara-Chow Hamamatsu, 433 Japan

Ultrahigh-resolution scanning electron microscopy of biological materials.

The age of ultrahigh-resolution scanning electron microscopy (SEM) began in 1985, when the UHS-T1, with a resolution of 0.5 nm, was developed. Commerc...
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