Nuclear Instruments and Methods in Physics Research B 269 (2011) 2251–2253

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Nuclear Instruments and Methods in Physics Research B

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Highly sensitive molecular detection with swift heavy ions

Yoshinobu Wakamatsu a,⇑, Hideaki Yamada a, Satoshi Ninomiya b,d, Brian N. Jones e, Toshio Seki a,d,Takaaki Aoki c,d, Roger Webb e, Jiro Matsuo b,d

a Department of Nuclear Engineering, Kyoto University, Sakyo, Kyoto 606-8501, Japanb Quantum Science and Engineering Center, Kyoto University, Uji, Kyoto 611-0011, Japanc Department of Electronic Science and Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japand Japan Science and Technology Agency (JST), Chiyoda, Kyoto 102-0075, Japane Ion Beam Centre, University of Surrey, Surrey GU2 7XH, UK

a r t i c l e i n f o

Article history:Available online 27 February 2011

Keywords:SIMSSecondary ion emissionSwift heavy ionElectronic sputteringBiomolecules

0168-583X/$ - see front matter � 2011 Published bydoi:10.1016/j.nimb.2011.02.069

⇑ Corresponding author.E-mail address: yoshinobu.w@nucleng.kyoto-u.ac.

a b s t r a c t

Various imaging techniques using microbeam have been applied in biology. Secondary ion mass spec-trometry (SIMS) is one of the prominent tools for biological imaging; SIMS can provide data on moleculardistribution in biological samples smaller than 1 lm. However, conventional SIMS has only low sensitiv-ity for molecular ions; therefore there is a need for beams of more sensitive primary ions. Plasma desorp-tion mass spectrometry (PDMS) is a method using high energy fission fragments from excitation of a 252Cfsource, and it allows ionization of large molecules (typically up to 20 kDa) due to the dense electronicexcitation. Although PDMS is not in use today because of the development of soft ionization methods,ionization induced by high energy ion collision still remains the only method which combines high spa-tial resolution and sensitive detection of large molecules. In this work, the secondary ion yield of aminoacid and phospholipid was measured for 6 MeV Cu4+. The yields were compared to bismuth cluster ions,which achieve relatively high yield. It was confirmed that the swift heavy ion has a couple of hundredtimes higher yield for large molecules than bismuth cluster ions.

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1. Introduction

Various microbeam techniques have been recently applied forbiological imaging. For example, focused ion beam and nuclearmicroprobe are used and elemental imaging in single cells was per-formed [1]. Although the techniques provide elemental mapping inbiological samples, knowledge of molecular distribution in organ-isms such as cells or tissues is still insufficient, because the organ-ism is composed of various kinds of organic molecules such aspeptides, hormones and lipids. Therefore, better molecular imagingtechniques are desired in biology. For the last decade secondary ionmass spectrometry (SIMS) has been applied in molecular imagingof biological samples [2–6]. SIMS allows analysis of both elementsand molecules and has been a powerful tool for biological imaging.Conventional SIMS however, has low sensitivity for high-massmolecules because of induced fragmentation, and therefore SIMSimaging has been limited to small molecules. SIMS using clusterincident ions such as bismuth cluster or fullerene achieved rela-tively high sensitivity for molecular ions of a couple of hundredsDa [7,8]. Sensitivity for higher mass ions, however, is still quitelow.

Elsevier B.V.

jp (Y. Wakamatsu).

Plasma desorption mass spectrometry (PDMS), which was re-ported in 1974, by Macfarene, is known as a mass spectrometrytechnique for large organic molecules [9]. In 252Cf-PDMS, high en-ergy fission fragments from a 252Cf source bombard and efficientlyionize atoms or molecules in solid samples. The energy of the fis-sion fragments of 252Cf is several tens of MeV. The high energyfragments induce dense electronic excitation in the target and ion-ization of intact molecules [10,11]. In the 1980s, PDMS was usedfor analysis of large organic molecules up to 20 kDa. After soft ion-ization techniques such as matrix-assisted laser desorption/ioniza-tion (MALDI) and electrospray ionization (ESI) became available inthe later 1980s, these methods have replaced PDMS [12,13]. Nev-ertheless, ionization induced by high energy ion collision has anadvantage in high spatial resolution. High energy ions from anaccelerator can be focused on the surface of biological samples.When the incident ions collide with the surface of samples, sec-ondary ions are emitted from a much narrower area (less than sev-eral tens nm2), compared to the other soft ionization methods.Therefore, we applied 6 MeV Cu4+ from a tandem accelerator forimaging, a method which we termed MeV-SIMS [14–16].

To demonstrate the advantage of MeV-SIMS over other meth-ods, we measured the secondary ion yields, i.e. the number of sec-ondary ions per incident primary ions, for several kinds ofmolecules for 25 keV Biþ3 and 6 MeV Cu4+. Because of the maximum

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primary ion dose density in static SIMS, which is called static limitand is typically 1012 ions/cm2, the number of the detected ions perunit area is limited by the secondary ion yield. In order to detect asignal from a molecule specie in 1 lm2 area, a secondary ion yieldhigher than 10�4 is required, for example. Therefore, the secondaryion yield is directly related to the highest spatial resolution achiev-able in SIMS imaging. Although the bismuth cluster ion is known asa primary ion giving a high secondary ion yield, imaging of largemolecules is difficult because of its low sensitivity. Arginine(C6H16N4O2) and distearoyl-phosphatidylcholine (DSPC, C44H88-NO8P) were chosen as two samples with different nominal mass,174.1 and 789.6 Da, respectively.

Fig. 2. SIMS spectra of arginine obtained with 25 keV Biþ3 and 6 MeV Cu4+. Thesecondary ion yields were 5.9 � 10�3 for 25 keV Biþ3 and 1.4 � 10�2 for 6 MeV Cu4+.

2. Experimental

The arginine and DSPC spin-coated thin firms were prepared asfollows. A DSPC chloroform solution of 10 ll was spin-coated onthe silicon wafer at 5000 rpm for 30 s. For the arginine aqueoussolution 10 ll was spin-coated on a washed silicon wafer at5000 rpm for 60 s.

SIMS measurements using 25 keV Biþ3 were performed by usingthe time-of-flight (TOF) SIMS system (TOF-SIMS 5, ION-TOF Inc.,Germany) at the University of Surrey.

SIMS measurements using 6 MeV Cu4+ were performed with theinstrument depicted schematically in Fig. 1. The samples were irra-diated with primary ions generated from the tandem accelerator atKyoto University. Secondary ions were analyzed with an orthogo-nal acceleration TOF (oa-TOF) mass spectrometer [17]. Secondaryions emitted from the sample were introduced into the quadrupoleion guide section and focused with a radio frequency (RF) voltageapplied. In the ion guide, the ions were cooled by collisions withhelium and transported to a microchannel plate (MCP). The TOFmeasurement was performed between the push-out-plate andMCP. The vacuum in the TOF analyzer was �10�5 Pa and wasachieved with a differential pumping system. The sample was keptin low vacuum (�103 Pa) with an orifice connecting the sampleand the ion guide sections.

3. Results and discussion

Fig. 2 shows the SIMS spectra of arginine using 25 keV Biþ3 and6 MeV Cu4+. In both spectra, the signals of protonated arginine areobserved at m/z = 175.1. The ions below m/z = 70 are not found inFig. 2b because of the cut-off effect from the quadrupole ion guide.The yield with 25 keV Biþ3 was 5.9 � 10�3, and the yield with 6 MeV

Fig. 1. Schematic diagram of SIMS instrument using 6 MeV Cu4+.

Cu4+ was 1.4 � 10�2 which is 2.4 times higher than with bismuthcluster ions. Therefore, both 25 keV Biþ3 and 6 MeV Cu4+ have suffi-cient yield in SIMS imaging for low-mass molecules. Although theyield for 6 MeV Cu4+ is higher than for bismuth cluster ions, the lat-ter one is also sufficiently high.

Fig. 3. SIMS spectra of DSPC for 25 keV Biþ3 and 6 MeV Cu4+. The secondary ionyields of 25 keV Biþ3 and 6 MeV Cu4+ were 2.1 � 10�5 and 8.9 � 10�3, respectively.

Table 1Comparison of secondary ion yield for protonated arginine (m/z = 175.1), protonatedphosphocholine headgroup (m/z = 184.1), and protonated DSPC (m/z = 790.6) forseveral primary ions.

Secondary ion Secondary ion yield, of

25 keV Biþ3 6 MeV Cu4+

Arginine + H+ (m/z = 175.1) 5.9 � 10�3 1.4 � 10�2

PC headgroup + H+ (m/z = 184.1) 6.3 � 10�3 5.4 � 10�2

DSPC + H+ (m/z = 790.6) 2.1 � 10�5 8.9 � 10�3

Y. Wakamatsu et al. / Nuclear Instruments and Methods in Physics Research B 269 (2011) 2251–2253 2253

Fig. 3a presents the spectrum of DSPC using 25 keV Biþ3 . Thepeak of m/z = 184.1 represents the protonated phosphocholine(PC) headgroup and m/z = 790.6 represents the molecular ions ofDSPC. In the spectrum of Biþ3 (Fig. 3a), the peak of m/z = 184.1 is rel-atively high, but at m/z = 790.6 the peak of molecular ions in thespectrum is quite low. In contrast, in Fig. 3b an intense peak wasobserved at m/z 790.6. The yields of the molecular ions with25 keV Biþ3 and 6 MeV Cu4+ were 2.1 � 10�5 and 8.9 � 10�3, respec-tively. The yield with 6 MeV Cu4+ was about two hundred timeshigher than that with 25 keV Biþ3 . Therefore, imaging of high-massmolecules is possible by using high energy ions. On the other hand,the dominant fragment peak from DSPC molecules at m/z = 184.1has a high secondary ion yield using either 25 keV Biþ3 or 6 MeVCu4+. Similar to arginine, the yield enhancement with 6 MeV Cu4+

compared to 25 keV Biþ3 was one order of magnitude only. The re-sults of the secondary ion yield measurement showed that swiftheavy ions have a higher sensitivity for large molecules than keVenergy ions. While the peak at m/z = 184.1 is observed in DSPC,the PC headgroup is detected in many similar phospholipids, forexample, DPPC [18]. For the study of the metabolism, it is consid-ered that the separation between similar phospholipids is veryimportant. Thus, the fact that mapping of the individual phospho-lipids is possible is important for future use.

In metal cluster ion bombardment, a much higher proportion oflow-mass molecules (less than a couple of hundreds Da) is pro-duced compared with monomer ions such as Ga+ [19]. Clusterion bombardment induces dense collision cascades which in turngenerate low mass ions [20]. However, for larger molecules, theamount of secondary ions created in a collision cascade is extre-mely limited. Instead, collision cascades induce fragmentations ofthe large molecules and only stable fragment ions are emitted fromthe sample surface.

In contrast to low energy primary ions that produce secondaryions only by collision cascades, high energy primary ions producethem by both electronic excitation and collision cascades. How-ever, since both electronic excitation and collision cascades effec-tively produce low mass secondary ions, the enhancement forlow mass ions is relatively low. As shown in Table. 1, similarenhancements were found for low mass molecular ions and frag-ments from large molecules. On the other hand, larger molecularions are dominantly derived from electronic excitation. Electronicexcitation generates delta electrons in the infratrack and theseelectrons induce the ejection of intact molecules from the outer re-gion, which is called ultratrack. Therefore, the secondary ion yieldincrease corresponds to the electronic stopping power increase[14,21]. When using 6 MeV Cu4+, the secondary ion yield is

enhanced by a couple of hundred times for larger molecules, be-cause the emission from the ultratrack, i.e. emission by electronicexcitation, is dominant for larger molecules. Therefore, the yieldenhancement increases particularly for higher mass molecules,giving swift heavy ions an advantage for SIMS of larger molecules.

4. Conclusion

As could be shown, the 6 MeV Cu4+ ions lead to be a 400 timeshigher secondary ion yield for high-mass molecules and 2.4 timeshigher yield for relatively low-mass molecules compared to bis-muth cluster incident ions. SIMS using high energy ions such as6 MeV Cu4+ achieves a high yield for molecules. This yield enhance-ment increases especially for heavy molecules and therefore, MeV-SIMS provides the possibility to visualize heavier molecules suchas phospholipids or peptides with high spatial resolution whichcannot be achieved in conventional SIMS.

Acknowledgements

The author also wishes to thank Prof. A. Ito, Prof. H. Shibata, andProf. H. Tsuchida for their advice, and Prof J. Watts, Dr. S. Hinder, K.Norizawa, M. Naito, M. Hada, K. Ichiki, S. Ibuki and Y. Yamamoto fortheir technical support.

This work was partially supported by the Core Research of Evo-lutional Science and Technology (CREST) of Japan Science andTechnology Agency.

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