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Atomic level analysis of biomolecules by the scanning atom probe
Applied Surface Science 256 (2009) 1210–1213
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Atomic level analysis of biomolecules by the scanning atom probe Osamu Nishikawa a,*, Masahiro Taniguchi a, Atsushi Ikai b a b
Department of Chemistry and Biology, Kanazawa Institute of Technology, 7-1 Ohgigaoka, Nonoichi 921-8501, Japan Laboratory of Biodynamics, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 6 June 2009
Utilizing the unique features of the scanning atom probe (SAP) the binding states of the biomolecules, leucine and methionine, are investigated at atomic level. The molecules are mass analyzed by detecting a single atom and/or clustering atoms field evaporated from a specimen surface. Since the field evaporation is a static process, the evaporated clustering atoms are closely related with the binding between atoms forming the molecules. For example, many thiophene radicals are detected when polythiophene is mass analyzed by the SAP. In the present study the specimens are prepared by immersing a micro cotton ball of single walled carbon nanotubes (SWCNT) in the leucine or methionine solution. The mass spectra obtained by analyzing the cotton balls exhibit singly and doubly ionized carbon ions of SWCNT and the characteristic fragments of the molecules, CH3, CHCH3, C4H7, CHNH2 and COOH for leucine and CH3, SCH3, C2H4, C4H7, CHNH2 and COOH for methionine. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Scanning atom probe Single walled carbon nanotubes Field evaporation Fragment ions Leucine Methionine
1. Introduction Since the introduction of the atom probe (AP) [1,2], the combined instrument of a field ion microscope with atomic resolution and a mass analyzer with the sensitivity of detecting a single ion, various metallic and semiconductor specimens have been mass analyzed at atomic level [3–5]. However, organic molecules and polymers are not analyzed extensively because the preparation of a suitable specimen for AP, a sharp needle, is impeding the analysis of these specimens. In order to remove this barrier a small funnel-shaped extraction electrode was introduced in the conventional AP [6,7]. The electrode confines the high field required for the field evaporation of surface atoms in a small space between an apex of a minute protrusion on a flat specimen and an open hole at the sharp end of the electrode. The electrode can scan over a specimen surface as a scanning tunnelling microscope (STM) [8] and stay over the apex of a protrusion. This type of the AP is named as the scanning atom probe (SAP). The extraction electrode allows to mass analyze a soft polythiophene film [9]. The obtained mass spectra have a sharp mass peak at the mass of thiophene radical SC4H2. Furthermore, no isolated sulphur atom is detected and most boron atoms doped in the polythiophene are bound with SC clusters. Similarly, the mass spectra of tetra-N-butylammonium hydroxide (TBA) show the characteristic clusters such as NC4H9, NC8H18 and NC12H27. No
* Corresponding author. Tel.: +81 76 248 9473; fax: +81 76 2946717. E-mail address: [email protected] (O. Nishikawa). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.05.154
isolated nitrogen atom was detected [10]. On the other hand nondirectionally and uniformly bound metal atoms are field evaporated one atom by one atom. Similarly, it has been also found that most carbon atoms of a clean nanotube are detected as a single atom ion [11]. This implies that the mass spectra of the nanotubes and those of the molecules can be separated. Accordingly, the specimens for the present study are prepared by immersing a micro cotton ball of single walled carbon nanotubes (SWCNT) in the solution of essential amino acids such as leucine, HO2CCH(NH2)CH2CH(CH3)2, and methionine, HO2CCH(NH2)CH2CH2SCH3. Since the introduction of Matrix Assisted Laser Desorption/ Ionization (MALDI) [12] the mass analysis of biomolecules are widely practised. However, no analyzed data is available for small molecules. Accordingly, the present study is the first attempt to mass analyze the small molecules at atomic level. 2. Experimental The structure of the SAP is described elsewhere [7]. The SAP mass analysis is proceeded by applying a positive pulsed voltage VP of 5 ns wide and a superposing positive DC voltage VDC to a specimen. The ratio of VP to (VDC + VP) is set at 20%. Flight times of the field evaporated ions are measured by the timer with 1 ns accuracy. Accordingly, the mass resolution m/Dm of the SAP is better than 1000. Since specimen atoms are field evaporated by nanosecond voltage pulses, conductive specimens are desirable for the SAP analysis. However, biomolecules are not conductive. Thus, the molecules are deposited on a conductive substrate. In order to avoid the catalytic reaction of metals and semiconductors, carbon
O. Nishikawa et al. / Applied Surface Science 256 (2009) 1210–1213
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Fig. 1. Mass spectrum of SWCNT. A number beside an upward arrow indicates the number of detected ions. The mass peak C+/C22+ is formed by C+ ions and C22+ ions. More than 80% of detected ions are C2+ and C+/C22+. The ratio of C2+ to C+/C22+ is close to 3 to 1. Few H ions are detected.
is assumed to be a suitable substrate. However, it has been found that most carbon nanotubes and graphite fibers are heavily contaminated and contain a significant amount of H and O forming various C–H, C–O, C–H–O clusters [11,13]. Some of these clusters are indistinguishable from the radicals constructing the molecules such as CH3 and COOH. Deletion of the characteristics mass peaks of the carbon substrate from the mass spectrum obtained by analyzing biomolecules is annoying because these carbon substrates neither have a uniform composition nor reproduce identical mass spectra. SAP analysis indicates that the single walled carbon nanotube (SWCNT) produced by the high pressure carbon monoxide process [14] exhibits a fairly simple and clean spectrum [12]. The largest mass peaks of the SWCNT are C2+ and C+/C22+, Fig. 1. Most other mass peaks are doubly charged carbon clusters with less than 1% of hydrogen and 2–3% of oxygen. A cotton ball of tangled long SWCNTs is silver pasted on a W tip. Then, the tip is dipped in a solution of sample molecules. The sample solutions contain 47 mg/ml leucine and 52 mg/ml methionine in 0.1 M HCl, respectively. The molecules are held between SWCNT fibers. 3. Results The SAP analysis proceeds by field evaporating SWCNTs and molecules from the surface layers to inside. Although the SWCNT
and molecule zones are randomly mixed, the SWCNT zone can be separated from the molecules zone by plotting the ions in the detection sequence. When the SWCNT zone is analyzed, most detected ions are singly and doubly charged carbon ions. In the molecule zone the number of the carbon ions is small and the number of other ions increases. Fig. 2 shows the mass spectrum obtained by analyzing the leucine deposited SWCNT. The detection of a large number of hydrogen ions may be due to the dissociation of the molecules and a long tail toward the larger mass side may be also due to the dissociation of the weak C–H bonds [7]. The detection sequence of the ions, Fig. 3, shows that most ions detected up to the 3850th ions are carbon. Thus, this is the SWCNT zone. After the 3850th ion the detection rate of other fragment ions increases indicating the leucine zone. The mass spectrum of the leucine zone exhibits the characteristic fragments such as CH3, CHCH3, C4H7, CHNH2 and COOH, Fig. 4. A large number of sodium ions are detected in the molecule zone. The origin of sodium ions is not known at present. The mass peaks of COOH and CH3 in Fig. 4 are comparably high. However, the mass peak of CH-CH3 is significantly small. Some of the fragment might be further dissociated into H and C. Fig. 5 is the mass spectrum of the SWCNT with methionine. Several mass peaks have a long tail toward the larger mass side as shown in Fig. 2. The detection sequence of this analysis,
Fig. 2. Mass spectrum of leucine deposited SWCNT. Most ions are carbon clusters of SWCNT. However, many H+ ions and other cluster ions are also detected.
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Fig. 3. Detection sequence of ions. The SWCNT zone and the leucine zone are clearly separated. The axis of ordinates is the number of designated ions and that of abscissas is the total number of detected ions. The vertical dotted line is the boundary of the SWCNT zone and the leucine zone.
Fig. 6, indicates that the SWCNT zone is up to 4300th ions and the methionine zone follows. The mass spectrum of the methionine zone, Fig. 7, shows that the characteristic fragments are CH3, C2H4, C4H7, CHNH2, COOH and SCH3. The mass peak designated as COOH is too large comparing with other mass peaks. This mass peak might be a joint mass peak of COOH and CSH. A large number of sodium ions is detected. Note that the detection rate decreases with depth. It may imply that the sparsely distributed sodium atoms in the molecule zone may migrate to the surface area by the applied field. Unexpected finding is that the fragment C4H7 is doubly charged as shown in Figs. 4 and 6. The mass peak of COOH in Fig. 7 is significantly larger than other mass peaks. On the other hand the mass peaks assumed to be related with sulphur such as CSH and CS can be designated as COOH and CO2. In order to raise the reliability of the present study, it is necessary to identify the detected ions accurately. The introduction of a mass analyzer with a higher mass resolution m/Dm better than 5000 is an indispensable requirement.
Fig. 4. Mass spectrum of the leucine zone. The mass peaks of the characteristic fragments are pointed by arrows. The number of ions plotted in this mass spectrum is 25.6% of the total number of ions shown in Fig. 2. However, the number of H+ plotted in this mass spectrum is 73.7%, CH3+ is 82.0%, C4H72+ is 100%, C2H4+ is 47%, CNH3+ is 68,6% and COOH+ is 87.0%. On the other hand, the number of C2+ is 6.1%, C+/C22+ is 6.9%, C2+/C42+ is 11.9% and C52+ is 8.6%. 81.1% of Na+ is plotted.
Fig. 5. Mass spectrum of methionine deposited SWCNT. H+ mass peak is large. The ratio of the number of C2+ and that of C+/C22+ is nearly equal.
O. Nishikawa et al. / Applied Surface Science 256 (2009) 1210–1213
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4. Conclusion This study is the first attempt to employ the SAP for the mass analysis of biomolecules and successfully demonstrates that the characteristic fragments can be obtained by the analysis. However, the abundance of the fragments does not agree well with the expected numbers. The detailed counting of the number of sulphur atoms and various fragments is required to elevate the reliability of the SAP analysis. Acknowledgements The authors deeply acknowledge Dr. R.H. Hauge of Carbon Nanotechnology Laboratory of Rice University for supplying the SWCNTs. This research is supported by the Japan Society for the Promotion of Science. References Fig. 6. Detection sequence of designated ions. The vertical dotted line is the boundary between the SWCNT zone and the methionine zone.
Fig. 7. Mass spectrum of the methionine zone. The number of ions of this mass spectrum is 39.6% of Fig. 5. However, the number of H+ is 78.6%, CH3+ is 97.0%, SCH3+ is 94.0%, C2H4+ is 67.6%, C4H72+ is 100%, CNH3+ is 97.3% and COOH+ is 99.4%. 94.3% of Na+ is plotted.
[1] E.W. Mu¨ller, J.A. Panitz, S.B. McLane, Rev. Sci. Instrum. 39 (1968) 83. [2] O. Nishikawa, K. Kurihara, M. Nachi, M. Konishi, M. Wada, Rev. Sci. Instrum. 52 (1981) 810. [3] A. Cerezo, G.D.W. Smith, Field ion microscopy: atom probe microanalysis, Encyclopaedia of Materials, vol. 4, Elsevier, Oxford, 2001. [4] O. Nishikawa, O. Kaneda, M. Shibata, E. Nomura, Phys. Rev. Lett. 53 (1984) 1252. [5] O. Nishikawa, T. Murakami, M. Watanabe, M. Taniguchi, T. Kuzumaki, S. Kondo, Jpn. J. Appl. Phys. 42 (2003) 4816. [6] O. Nishikawa, M. Kimoto, Appl. Surf. Sci. 76/77 (1994) 424. [7] O. Nishikawa, Y. Ohtani, K. Maeda, M. Watanabe, K. Tanaka, Mater. Charact. 44 (2000) 29. [8] G. Binnig, H. Rohrer, Ch. Gerber, E. Weibel, Phys. Rev. Lett. 50 (1983) 120. [9] O. Nishikawa, M. Taniguchi, Chin. J. Phys. 43 (No. I–II) (2006) 111. [10] O. Nishikawa, M. Taniguchi, S. Watanabe, A. Yamagishi, T. Sasaki, Jpn. J. Appl. Phys. 45 (2006) 1892. [11] O. Nishikawa, M. Taniguchi, Y. Saito, J. Vac. Sci. Technol., A 26 (2008) 1074. [12] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [13] O. Nishikawa, M. Taniguchi, M. Ushirozawa, J. Vac. Sci. Technol., B 26 (2008) 735. [14] M.J. Bronikowski, P.A. Willis, D.T. Colbert, K.A. Smith, R.E. Smalley, J. Vac. Sci. Technol., A 18 (2001) 1800.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Atomic level analysis of biomolecules by the scanning atom probe Osamu Nishikawa a,*, Masahiro Taniguchi a, Atsushi Ikai b a b
Department of Chemistry and Biology, Kanazawa Institute of Technology, 7-1 Ohgigaoka, Nonoichi 921-8501, Japan Laboratory of Biodynamics, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 6 June 2009
Utilizing the unique features of the scanning atom probe (SAP) the binding states of the biomolecules, leucine and methionine, are investigated at atomic level. The molecules are mass analyzed by detecting a single atom and/or clustering atoms field evaporated from a specimen surface. Since the field evaporation is a static process, the evaporated clustering atoms are closely related with the binding between atoms forming the molecules. For example, many thiophene radicals are detected when polythiophene is mass analyzed by the SAP. In the present study the specimens are prepared by immersing a micro cotton ball of single walled carbon nanotubes (SWCNT) in the leucine or methionine solution. The mass spectra obtained by analyzing the cotton balls exhibit singly and doubly ionized carbon ions of SWCNT and the characteristic fragments of the molecules, CH3, CHCH3, C4H7, CHNH2 and COOH for leucine and CH3, SCH3, C2H4, C4H7, CHNH2 and COOH for methionine. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Scanning atom probe Single walled carbon nanotubes Field evaporation Fragment ions Leucine Methionine
1. Introduction Since the introduction of the atom probe (AP) [1,2], the combined instrument of a field ion microscope with atomic resolution and a mass analyzer with the sensitivity of detecting a single ion, various metallic and semiconductor specimens have been mass analyzed at atomic level [3–5]. However, organic molecules and polymers are not analyzed extensively because the preparation of a suitable specimen for AP, a sharp needle, is impeding the analysis of these specimens. In order to remove this barrier a small funnel-shaped extraction electrode was introduced in the conventional AP [6,7]. The electrode confines the high field required for the field evaporation of surface atoms in a small space between an apex of a minute protrusion on a flat specimen and an open hole at the sharp end of the electrode. The electrode can scan over a specimen surface as a scanning tunnelling microscope (STM) [8] and stay over the apex of a protrusion. This type of the AP is named as the scanning atom probe (SAP). The extraction electrode allows to mass analyze a soft polythiophene film [9]. The obtained mass spectra have a sharp mass peak at the mass of thiophene radical SC4H2. Furthermore, no isolated sulphur atom is detected and most boron atoms doped in the polythiophene are bound with SC clusters. Similarly, the mass spectra of tetra-N-butylammonium hydroxide (TBA) show the characteristic clusters such as NC4H9, NC8H18 and NC12H27. No
* Corresponding author. Tel.: +81 76 248 9473; fax: +81 76 2946717. E-mail address: [email protected] (O. Nishikawa). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.05.154
isolated nitrogen atom was detected [10]. On the other hand nondirectionally and uniformly bound metal atoms are field evaporated one atom by one atom. Similarly, it has been also found that most carbon atoms of a clean nanotube are detected as a single atom ion [11]. This implies that the mass spectra of the nanotubes and those of the molecules can be separated. Accordingly, the specimens for the present study are prepared by immersing a micro cotton ball of single walled carbon nanotubes (SWCNT) in the solution of essential amino acids such as leucine, HO2CCH(NH2)CH2CH(CH3)2, and methionine, HO2CCH(NH2)CH2CH2SCH3. Since the introduction of Matrix Assisted Laser Desorption/ Ionization (MALDI) [12] the mass analysis of biomolecules are widely practised. However, no analyzed data is available for small molecules. Accordingly, the present study is the first attempt to mass analyze the small molecules at atomic level. 2. Experimental The structure of the SAP is described elsewhere [7]. The SAP mass analysis is proceeded by applying a positive pulsed voltage VP of 5 ns wide and a superposing positive DC voltage VDC to a specimen. The ratio of VP to (VDC + VP) is set at 20%. Flight times of the field evaporated ions are measured by the timer with 1 ns accuracy. Accordingly, the mass resolution m/Dm of the SAP is better than 1000. Since specimen atoms are field evaporated by nanosecond voltage pulses, conductive specimens are desirable for the SAP analysis. However, biomolecules are not conductive. Thus, the molecules are deposited on a conductive substrate. In order to avoid the catalytic reaction of metals and semiconductors, carbon
O. Nishikawa et al. / Applied Surface Science 256 (2009) 1210–1213
1211
Fig. 1. Mass spectrum of SWCNT. A number beside an upward arrow indicates the number of detected ions. The mass peak C+/C22+ is formed by C+ ions and C22+ ions. More than 80% of detected ions are C2+ and C+/C22+. The ratio of C2+ to C+/C22+ is close to 3 to 1. Few H ions are detected.
is assumed to be a suitable substrate. However, it has been found that most carbon nanotubes and graphite fibers are heavily contaminated and contain a significant amount of H and O forming various C–H, C–O, C–H–O clusters [11,13]. Some of these clusters are indistinguishable from the radicals constructing the molecules such as CH3 and COOH. Deletion of the characteristics mass peaks of the carbon substrate from the mass spectrum obtained by analyzing biomolecules is annoying because these carbon substrates neither have a uniform composition nor reproduce identical mass spectra. SAP analysis indicates that the single walled carbon nanotube (SWCNT) produced by the high pressure carbon monoxide process [14] exhibits a fairly simple and clean spectrum [12]. The largest mass peaks of the SWCNT are C2+ and C+/C22+, Fig. 1. Most other mass peaks are doubly charged carbon clusters with less than 1% of hydrogen and 2–3% of oxygen. A cotton ball of tangled long SWCNTs is silver pasted on a W tip. Then, the tip is dipped in a solution of sample molecules. The sample solutions contain 47 mg/ml leucine and 52 mg/ml methionine in 0.1 M HCl, respectively. The molecules are held between SWCNT fibers. 3. Results The SAP analysis proceeds by field evaporating SWCNTs and molecules from the surface layers to inside. Although the SWCNT
and molecule zones are randomly mixed, the SWCNT zone can be separated from the molecules zone by plotting the ions in the detection sequence. When the SWCNT zone is analyzed, most detected ions are singly and doubly charged carbon ions. In the molecule zone the number of the carbon ions is small and the number of other ions increases. Fig. 2 shows the mass spectrum obtained by analyzing the leucine deposited SWCNT. The detection of a large number of hydrogen ions may be due to the dissociation of the molecules and a long tail toward the larger mass side may be also due to the dissociation of the weak C–H bonds [7]. The detection sequence of the ions, Fig. 3, shows that most ions detected up to the 3850th ions are carbon. Thus, this is the SWCNT zone. After the 3850th ion the detection rate of other fragment ions increases indicating the leucine zone. The mass spectrum of the leucine zone exhibits the characteristic fragments such as CH3, CHCH3, C4H7, CHNH2 and COOH, Fig. 4. A large number of sodium ions are detected in the molecule zone. The origin of sodium ions is not known at present. The mass peaks of COOH and CH3 in Fig. 4 are comparably high. However, the mass peak of CH-CH3 is significantly small. Some of the fragment might be further dissociated into H and C. Fig. 5 is the mass spectrum of the SWCNT with methionine. Several mass peaks have a long tail toward the larger mass side as shown in Fig. 2. The detection sequence of this analysis,
Fig. 2. Mass spectrum of leucine deposited SWCNT. Most ions are carbon clusters of SWCNT. However, many H+ ions and other cluster ions are also detected.
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Fig. 3. Detection sequence of ions. The SWCNT zone and the leucine zone are clearly separated. The axis of ordinates is the number of designated ions and that of abscissas is the total number of detected ions. The vertical dotted line is the boundary of the SWCNT zone and the leucine zone.
Fig. 6, indicates that the SWCNT zone is up to 4300th ions and the methionine zone follows. The mass spectrum of the methionine zone, Fig. 7, shows that the characteristic fragments are CH3, C2H4, C4H7, CHNH2, COOH and SCH3. The mass peak designated as COOH is too large comparing with other mass peaks. This mass peak might be a joint mass peak of COOH and CSH. A large number of sodium ions is detected. Note that the detection rate decreases with depth. It may imply that the sparsely distributed sodium atoms in the molecule zone may migrate to the surface area by the applied field. Unexpected finding is that the fragment C4H7 is doubly charged as shown in Figs. 4 and 6. The mass peak of COOH in Fig. 7 is significantly larger than other mass peaks. On the other hand the mass peaks assumed to be related with sulphur such as CSH and CS can be designated as COOH and CO2. In order to raise the reliability of the present study, it is necessary to identify the detected ions accurately. The introduction of a mass analyzer with a higher mass resolution m/Dm better than 5000 is an indispensable requirement.
Fig. 4. Mass spectrum of the leucine zone. The mass peaks of the characteristic fragments are pointed by arrows. The number of ions plotted in this mass spectrum is 25.6% of the total number of ions shown in Fig. 2. However, the number of H+ plotted in this mass spectrum is 73.7%, CH3+ is 82.0%, C4H72+ is 100%, C2H4+ is 47%, CNH3+ is 68,6% and COOH+ is 87.0%. On the other hand, the number of C2+ is 6.1%, C+/C22+ is 6.9%, C2+/C42+ is 11.9% and C52+ is 8.6%. 81.1% of Na+ is plotted.
Fig. 5. Mass spectrum of methionine deposited SWCNT. H+ mass peak is large. The ratio of the number of C2+ and that of C+/C22+ is nearly equal.
O. Nishikawa et al. / Applied Surface Science 256 (2009) 1210–1213
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4. Conclusion This study is the first attempt to employ the SAP for the mass analysis of biomolecules and successfully demonstrates that the characteristic fragments can be obtained by the analysis. However, the abundance of the fragments does not agree well with the expected numbers. The detailed counting of the number of sulphur atoms and various fragments is required to elevate the reliability of the SAP analysis. Acknowledgements The authors deeply acknowledge Dr. R.H. Hauge of Carbon Nanotechnology Laboratory of Rice University for supplying the SWCNTs. This research is supported by the Japan Society for the Promotion of Science. References Fig. 6. Detection sequence of designated ions. The vertical dotted line is the boundary between the SWCNT zone and the methionine zone.
Fig. 7. Mass spectrum of the methionine zone. The number of ions of this mass spectrum is 39.6% of Fig. 5. However, the number of H+ is 78.6%, CH3+ is 97.0%, SCH3+ is 94.0%, C2H4+ is 67.6%, C4H72+ is 100%, CNH3+ is 97.3% and COOH+ is 99.4%. 94.3% of Na+ is plotted.
[1] E.W. Mu¨ller, J.A. Panitz, S.B. McLane, Rev. Sci. Instrum. 39 (1968) 83. [2] O. Nishikawa, K. Kurihara, M. Nachi, M. Konishi, M. Wada, Rev. Sci. Instrum. 52 (1981) 810. [3] A. Cerezo, G.D.W. Smith, Field ion microscopy: atom probe microanalysis, Encyclopaedia of Materials, vol. 4, Elsevier, Oxford, 2001. [4] O. Nishikawa, O. Kaneda, M. Shibata, E. Nomura, Phys. Rev. Lett. 53 (1984) 1252. [5] O. Nishikawa, T. Murakami, M. Watanabe, M. Taniguchi, T. Kuzumaki, S. Kondo, Jpn. J. Appl. Phys. 42 (2003) 4816. [6] O. Nishikawa, M. Kimoto, Appl. Surf. Sci. 76/77 (1994) 424. [7] O. Nishikawa, Y. Ohtani, K. Maeda, M. Watanabe, K. Tanaka, Mater. Charact. 44 (2000) 29. [8] G. Binnig, H. Rohrer, Ch. Gerber, E. Weibel, Phys. Rev. Lett. 50 (1983) 120. [9] O. Nishikawa, M. Taniguchi, Chin. J. Phys. 43 (No. I–II) (2006) 111. [10] O. Nishikawa, M. Taniguchi, S. Watanabe, A. Yamagishi, T. Sasaki, Jpn. J. Appl. Phys. 45 (2006) 1892. [11] O. Nishikawa, M. Taniguchi, Y. Saito, J. Vac. Sci. Technol., A 26 (2008) 1074. [12] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [13] O. Nishikawa, M. Taniguchi, M. Ushirozawa, J. Vac. Sci. Technol., B 26 (2008) 735. [14] M.J. Bronikowski, P.A. Willis, D.T. Colbert, K.A. Smith, R.E. Smalley, J. Vac. Sci. Technol., A 18 (2001) 1800.