Arch.7 orul Biol. Vol. ?I. pp. 141 to 145. Pergamon
Press
1976. Printed in Great
Britain
SHORT COMMUNICATION ELECTRON
MICROSCOPY OF ION-SPUTTERED HUMAN ENAMEL
T. NAKATA, Y. TSUJI, J. FUJIWARA, K. YAMAMOTO and S. MIYOSHI Department of Oral Anatomy, Osaka University Dental School, Joancho-32, Kita-ku, Osaka 530, Japan
Summary-The
ultrastructure of human enamel was studied by ion-sputtering combined with scanning electron microscopy and 2000 kV and 100 kV transmission electron microscopy. The enamel prisms were not thinned uniformly but the prism sheath and some portions in the prism head were selectively eroded by the argon-ion beam. The periphery of the prisms was electron-lucent in 2000 kV transmission electron microscopy. However, with 100 kV transmission electron microscopy, crystals separated by 1.5-2 nm spaces were clearly seen. Various Moire patterns of parallel lines were observed on some crystals and ion-sputtering produced spotted defects on all crystals.
;.n recent years, ion-sputtering (ion-etching, ion-bombardment, ion-thinning, ion-polishing, ion-micromilling) has been applied to biological materials. Lewis, Osborn and Stuart (1968) and Fujita, Nagatani and Hattori (1974) observed the internal structure of ionatched erythrocytes and leucocytes and Hodges et al. (1972) examined the etched patterns of chick erythror:ytes and cultured mammalian cells with various ions (hydrogen, helium, argon, oxygen) by scanning electron microscopy (S.E.M.). In hard dental tissues, Boyde (1974) investigated icn-etched human dentine and Hamilton, Judd and Ansell (1973) and Orams et al. (1974) reported on etched enamel studied by transmission electron microscopy (T.E.M.). Thick sections, however, cannot be observed by the 100 kV T.E.M. although, as the accelerating voltage goes up, the penetrating power of electrons increases and allows thicker sections to be observed. Incisors from young adults were fixed immediately after extraction in 10 per cent neutral formalin for 48 hr and then sections 2-3 mm thick were cut longit ldinally with a dental diamond disc. The slices were ground to 5pm thick using Fremlin’s technique (i’remlin, Mathieson and Hardwick, 1961). The specimens were sandwiched in double grids and sputtered with a beam of ionized argon for 2448 hr by an ionstuttering instrument (IB-1 type, Shinko Seiki Co., Ltd. Kobe, Japan) using vacuum 5 x 10m4 torr in the etching chamber and an incident angle of beam to the specimen of 45”. Gun potentials were $6 kV and beam current 0.103 mA. The specimens were rotated mechanically by the gimbals and bombarded from each side to provide more uniform ion-bombardment and examined under the Hitachi HU-2000, HU-12 A (transmission type) and JEOL-JSM-U, (scanning type) at accelerating voltage 2000 kV, 100 kV and 25 kV respectively. Thick sections (about 2 pm thick) of enamel could be observed at 2000 kV. Prism sheaths sectioned
transversely and some parts of enamel prisms which were preferentially sputtered appeared to be electronlucent (Fig. 1). The crystals in 2pm thick sections were orientated in all directions and the difference of the crystal orientation between the head and tail regions was uncertain. With scanning electron microscopy, the surface of ion-sputtered enamel presented crater-like concave images and the prism sheaths were depressed (Fig. 2). With T.E.M., the electron-lucent portion was similar to the concave images seen with scanning electron microscopy. As ion-sputtering was continued, electron-lucent areas became larger and the centres of the head and tail regions of prisms remained unchanged (Fig. 3a). Thus the rate of destruction by sputtering varied in different parts of prisms. At higher magnification, the crystals were packed tightly and some crystals were electron dense. Many Moire patterns of parallel lines due to the duplication of crystals were clearly seen (Fig. 3b). The electron-lucent portion surrounding the prism head at 2000 kV was thin enough to observe by 100 kV transmission electron microscopy. The crystals of the translucent portion at 2000 kV were seen distinctly and were separated by spaces 1+2nm wide which may be filled with organic matrix. Dark or clear crystals were visible by 100 kV T.E.M. (Fig. 4). The crystals etched by argon-ion showed spotted defects and there were some pores which connected with the intercrystalline spaces (Fig. 4). The cause of the selective sputtering is still obscure. The factors affecting the sputtering ratio are considered to be the ion energy, incident angle of the ion beam to the specimen, atomic-binding energy on the specimen surface and orientation of the crystalline mineral components. Boyde and Stewart (1962a, 196213)studied the etching of dental hard tissues with scanning electron microscopy and reported that enamel tufts were eroded rapidly, that the dentine was etched more rapidly than the enamel and that the 141
142
T. Nakata, Y. Tsuji, J. Fujiwara, K. Yamamoto and S. Miyoshi
intertubular dentine quickly etched away compared with peritubular dentine. They also showed that differential etching occurred in the bulk of enamel and suggested that there is a variation of sputtering coefficient dependent on the orientation of hydroxyapatite crystals. In our study, the most readily sputtered portion was the sheaths and parts of the enamel prisms. The prism sheath is known to be rich in organic substance and may therefore be eroded quickly but the etched portion of the enamel prisms is highly calcified. Yoshida, Konishi and Nagata (1973) reported that the ion-sputtered surface of the stationary specimens of MnAl (r) alloy revealed the “arrow-feathered pattern” but, when specimens were rotated mechanically during the ion-etching, this pattern could be hardly recognized. Certainly the etching pattern of the stationary specimen may be influenced by crystal orientation but sections which are rotated mechanically by gimbals are considered to be uniformly sputtered and thinned with little regard to the orientation of the crystals. Other possibilities can be considered; for example the etched portion of enamel prisms may have a different chemical component compared with the remainder in addition to having a particular crystal orientation. It is said that hydroxyapatite crystals are orientated parallel to the long axes of the prisms in the upper part of the head region and gradually change in direction toward the tail region (Meckel, Griebstein and Neal, 1965). In 2pm thick sections, however many crystals were in layers; the difference of the crystal arrangement between the head and tail region was therefore indistinct. Scott, Simmelink and Nygaard (1974) reported that the peripheral regions of the enamel prisms remained intact in enamel etched by acid although the central portion of each crystal had been lost. However the ion-etched crystals in our study did not show the appearances described when crystals are treated by acid. Instead, the peripheral regions of the enamel prisms were eroded quickly and disappeared and every crystal showed spotted defects. The cause of the difference between the action by acid and argon-ion is unknown. Orams et al. (1974) examined ion-sputtered human enamel by 200 kV transmission electron microscopy and observed the electron-lucent channels or pores (l-10nm wide). They suggested that these channels contained soft material and would permit the passage of ions and small molecules. We noted a small number of the similar structures (Fig. 4). They extended to intercrystalline spaces and their
density was similar to that of these spaces. We suspect that this structure is a part of the intercrystalline space that is readily eroded by ion-sputtering.
Acknowledgmenr-This work supported by Grant 887073 in aid for Developmental Scientific Research of the Japanese Ministry of Education.
REFERENCE
Boyde A. and tissues with 159-172. Boyde A. and the erosion
Stewart A. D. G. 1962a. A study of dental argon ion beams. J. Ultrastruct Res. 7,
Stewart A. D. G. 1962b. Investigation of of tooth sections with an argon ion beam. Proc. 5th. Inter. Congr. Electron Microscopy, Philadelphia, 1962, 2, QQ-9, Academic Press, New York. Boyde A. 1974. Transmission electron microscopy of ion beam thinned dentine. Cell Tiss. Res. 152, 543-550. Fremlin J. H., Mathieson J. and Hardwick J. L. 1961. The preparation of thin sections of dental enamel. Archs oral Biol. 5, 55-60. Fujita T., Nagatani T. and Hattori A. 1974. A simple method of ion etching for biological material. An application to blood cells and spermatozoa. Archs histol. jap., 36, 195-204. Hamilton W. J. Jr., Judd G. and Ansell G. S. 1973. Ultrastructure of human enamel specimens prepared by ion micromilling. J. dent. Res. 52, 703-710. Hodges G. M., Muir M. D., Sella C. and Carteaud A. J. P. 1972. The effect of radio-frequency sputter ion etching and ion-beam etching on biological material: a scanning electron microscope study. J. Microsc. 95, 445-451. Lewis S. M., Osborn J. S. and Stuart P. R. 1968. Demonstration of an internal structure within the red blood cell by ion etching and scanning electron microscopy. Nature, 220, 614-616. Meckel A. H., Griebstein W. J. and Neal R. J. 1965. Structure of mature human dental enamel as observed by electron microscopy. Archs oral Biol. 10, 775-783. Orams H. J., Phakey W. A., Rachinger W. A. and Zybert J. J. 1974. Visualisation of micropore structure in human dental enamel. Nature, 252, 584585. Scott D. B., Simmelink J. W. and Nygaard V. 1974. Structural aspect of dental caries. J. dent. Rex 53, 161-178. Yoshida K., Konishi R. and Nagata S. 1973. Ion etching of MnAl(r) alloy and its application to thin foil preparation for transmission electron microscopy. J. electron Microsc. 22, 5-11.
Ion-sputtered
Plate
enamel
1 overleaf
143
144
T. Nakata, Y. Tsuji, J. Fujiwara, K. Yamamoto and S. miyoshi
Plate 1. Fig. 1. Transmission electron micrograph of ion-sputtered enamel at 2000 kV. The sheath appears to be electron-lucent and the prism head (PH) is partially removed by the argon ion-sputtering. x 4300 Fig. 2. Scanning electron micrograph of the surface of ion-sputtered enamel. The sheath is depressed and the surface of enamel has concave crater-like images. x 4300 Fig. 3a. Transmission electron micrograph of ion-sputtered enamel at 2000 kV. As the ion-sputtering was continued, the electron-lucent areas of enamel prisms became larger. x 4000 Fig. 3b. Higher magnification
of enamel crystals at 2000 kV. Arrows indicate MoirC patterns of parallel lines. x 45,000
Fig. 4. Enamel crystals by 100 kV transmission electron microscopy. The crystals are separated by intercrystalline space and have spotted defects. Arrows indicate electron-lucent channels. x 144,000
Ion-sputtered
enamel
Plate 1.
145
Press
1976. Printed in Great
Britain
SHORT COMMUNICATION ELECTRON
MICROSCOPY OF ION-SPUTTERED HUMAN ENAMEL
T. NAKATA, Y. TSUJI, J. FUJIWARA, K. YAMAMOTO and S. MIYOSHI Department of Oral Anatomy, Osaka University Dental School, Joancho-32, Kita-ku, Osaka 530, Japan
Summary-The
ultrastructure of human enamel was studied by ion-sputtering combined with scanning electron microscopy and 2000 kV and 100 kV transmission electron microscopy. The enamel prisms were not thinned uniformly but the prism sheath and some portions in the prism head were selectively eroded by the argon-ion beam. The periphery of the prisms was electron-lucent in 2000 kV transmission electron microscopy. However, with 100 kV transmission electron microscopy, crystals separated by 1.5-2 nm spaces were clearly seen. Various Moire patterns of parallel lines were observed on some crystals and ion-sputtering produced spotted defects on all crystals.
;.n recent years, ion-sputtering (ion-etching, ion-bombardment, ion-thinning, ion-polishing, ion-micromilling) has been applied to biological materials. Lewis, Osborn and Stuart (1968) and Fujita, Nagatani and Hattori (1974) observed the internal structure of ionatched erythrocytes and leucocytes and Hodges et al. (1972) examined the etched patterns of chick erythror:ytes and cultured mammalian cells with various ions (hydrogen, helium, argon, oxygen) by scanning electron microscopy (S.E.M.). In hard dental tissues, Boyde (1974) investigated icn-etched human dentine and Hamilton, Judd and Ansell (1973) and Orams et al. (1974) reported on etched enamel studied by transmission electron microscopy (T.E.M.). Thick sections, however, cannot be observed by the 100 kV T.E.M. although, as the accelerating voltage goes up, the penetrating power of electrons increases and allows thicker sections to be observed. Incisors from young adults were fixed immediately after extraction in 10 per cent neutral formalin for 48 hr and then sections 2-3 mm thick were cut longit ldinally with a dental diamond disc. The slices were ground to 5pm thick using Fremlin’s technique (i’remlin, Mathieson and Hardwick, 1961). The specimens were sandwiched in double grids and sputtered with a beam of ionized argon for 2448 hr by an ionstuttering instrument (IB-1 type, Shinko Seiki Co., Ltd. Kobe, Japan) using vacuum 5 x 10m4 torr in the etching chamber and an incident angle of beam to the specimen of 45”. Gun potentials were $6 kV and beam current 0.103 mA. The specimens were rotated mechanically by the gimbals and bombarded from each side to provide more uniform ion-bombardment and examined under the Hitachi HU-2000, HU-12 A (transmission type) and JEOL-JSM-U, (scanning type) at accelerating voltage 2000 kV, 100 kV and 25 kV respectively. Thick sections (about 2 pm thick) of enamel could be observed at 2000 kV. Prism sheaths sectioned
transversely and some parts of enamel prisms which were preferentially sputtered appeared to be electronlucent (Fig. 1). The crystals in 2pm thick sections were orientated in all directions and the difference of the crystal orientation between the head and tail regions was uncertain. With scanning electron microscopy, the surface of ion-sputtered enamel presented crater-like concave images and the prism sheaths were depressed (Fig. 2). With T.E.M., the electron-lucent portion was similar to the concave images seen with scanning electron microscopy. As ion-sputtering was continued, electron-lucent areas became larger and the centres of the head and tail regions of prisms remained unchanged (Fig. 3a). Thus the rate of destruction by sputtering varied in different parts of prisms. At higher magnification, the crystals were packed tightly and some crystals were electron dense. Many Moire patterns of parallel lines due to the duplication of crystals were clearly seen (Fig. 3b). The electron-lucent portion surrounding the prism head at 2000 kV was thin enough to observe by 100 kV transmission electron microscopy. The crystals of the translucent portion at 2000 kV were seen distinctly and were separated by spaces 1+2nm wide which may be filled with organic matrix. Dark or clear crystals were visible by 100 kV T.E.M. (Fig. 4). The crystals etched by argon-ion showed spotted defects and there were some pores which connected with the intercrystalline spaces (Fig. 4). The cause of the selective sputtering is still obscure. The factors affecting the sputtering ratio are considered to be the ion energy, incident angle of the ion beam to the specimen, atomic-binding energy on the specimen surface and orientation of the crystalline mineral components. Boyde and Stewart (1962a, 196213)studied the etching of dental hard tissues with scanning electron microscopy and reported that enamel tufts were eroded rapidly, that the dentine was etched more rapidly than the enamel and that the 141
142
T. Nakata, Y. Tsuji, J. Fujiwara, K. Yamamoto and S. Miyoshi
intertubular dentine quickly etched away compared with peritubular dentine. They also showed that differential etching occurred in the bulk of enamel and suggested that there is a variation of sputtering coefficient dependent on the orientation of hydroxyapatite crystals. In our study, the most readily sputtered portion was the sheaths and parts of the enamel prisms. The prism sheath is known to be rich in organic substance and may therefore be eroded quickly but the etched portion of the enamel prisms is highly calcified. Yoshida, Konishi and Nagata (1973) reported that the ion-sputtered surface of the stationary specimens of MnAl (r) alloy revealed the “arrow-feathered pattern” but, when specimens were rotated mechanically during the ion-etching, this pattern could be hardly recognized. Certainly the etching pattern of the stationary specimen may be influenced by crystal orientation but sections which are rotated mechanically by gimbals are considered to be uniformly sputtered and thinned with little regard to the orientation of the crystals. Other possibilities can be considered; for example the etched portion of enamel prisms may have a different chemical component compared with the remainder in addition to having a particular crystal orientation. It is said that hydroxyapatite crystals are orientated parallel to the long axes of the prisms in the upper part of the head region and gradually change in direction toward the tail region (Meckel, Griebstein and Neal, 1965). In 2pm thick sections, however many crystals were in layers; the difference of the crystal arrangement between the head and tail region was therefore indistinct. Scott, Simmelink and Nygaard (1974) reported that the peripheral regions of the enamel prisms remained intact in enamel etched by acid although the central portion of each crystal had been lost. However the ion-etched crystals in our study did not show the appearances described when crystals are treated by acid. Instead, the peripheral regions of the enamel prisms were eroded quickly and disappeared and every crystal showed spotted defects. The cause of the difference between the action by acid and argon-ion is unknown. Orams et al. (1974) examined ion-sputtered human enamel by 200 kV transmission electron microscopy and observed the electron-lucent channels or pores (l-10nm wide). They suggested that these channels contained soft material and would permit the passage of ions and small molecules. We noted a small number of the similar structures (Fig. 4). They extended to intercrystalline spaces and their
density was similar to that of these spaces. We suspect that this structure is a part of the intercrystalline space that is readily eroded by ion-sputtering.
Acknowledgmenr-This work supported by Grant 887073 in aid for Developmental Scientific Research of the Japanese Ministry of Education.
REFERENCE
Boyde A. and tissues with 159-172. Boyde A. and the erosion
Stewart A. D. G. 1962a. A study of dental argon ion beams. J. Ultrastruct Res. 7,
Stewart A. D. G. 1962b. Investigation of of tooth sections with an argon ion beam. Proc. 5th. Inter. Congr. Electron Microscopy, Philadelphia, 1962, 2, QQ-9, Academic Press, New York. Boyde A. 1974. Transmission electron microscopy of ion beam thinned dentine. Cell Tiss. Res. 152, 543-550. Fremlin J. H., Mathieson J. and Hardwick J. L. 1961. The preparation of thin sections of dental enamel. Archs oral Biol. 5, 55-60. Fujita T., Nagatani T. and Hattori A. 1974. A simple method of ion etching for biological material. An application to blood cells and spermatozoa. Archs histol. jap., 36, 195-204. Hamilton W. J. Jr., Judd G. and Ansell G. S. 1973. Ultrastructure of human enamel specimens prepared by ion micromilling. J. dent. Res. 52, 703-710. Hodges G. M., Muir M. D., Sella C. and Carteaud A. J. P. 1972. The effect of radio-frequency sputter ion etching and ion-beam etching on biological material: a scanning electron microscope study. J. Microsc. 95, 445-451. Lewis S. M., Osborn J. S. and Stuart P. R. 1968. Demonstration of an internal structure within the red blood cell by ion etching and scanning electron microscopy. Nature, 220, 614-616. Meckel A. H., Griebstein W. J. and Neal R. J. 1965. Structure of mature human dental enamel as observed by electron microscopy. Archs oral Biol. 10, 775-783. Orams H. J., Phakey W. A., Rachinger W. A. and Zybert J. J. 1974. Visualisation of micropore structure in human dental enamel. Nature, 252, 584585. Scott D. B., Simmelink J. W. and Nygaard V. 1974. Structural aspect of dental caries. J. dent. Rex 53, 161-178. Yoshida K., Konishi R. and Nagata S. 1973. Ion etching of MnAl(r) alloy and its application to thin foil preparation for transmission electron microscopy. J. electron Microsc. 22, 5-11.
Ion-sputtered
Plate
enamel
1 overleaf
143
144
T. Nakata, Y. Tsuji, J. Fujiwara, K. Yamamoto and S. miyoshi
Plate 1. Fig. 1. Transmission electron micrograph of ion-sputtered enamel at 2000 kV. The sheath appears to be electron-lucent and the prism head (PH) is partially removed by the argon ion-sputtering. x 4300 Fig. 2. Scanning electron micrograph of the surface of ion-sputtered enamel. The sheath is depressed and the surface of enamel has concave crater-like images. x 4300 Fig. 3a. Transmission electron micrograph of ion-sputtered enamel at 2000 kV. As the ion-sputtering was continued, the electron-lucent areas of enamel prisms became larger. x 4000 Fig. 3b. Higher magnification
of enamel crystals at 2000 kV. Arrows indicate MoirC patterns of parallel lines. x 45,000
Fig. 4. Enamel crystals by 100 kV transmission electron microscopy. The crystals are separated by intercrystalline space and have spotted defects. Arrows indicate electron-lucent channels. x 144,000
Ion-sputtered
enamel
Plate 1.
145
