363 SIMS Proceedings Papers Received: 29 September 2009 Revised: 19 February 2010 Accepted: 12 March 2010 Published online in Wiley Online Library: 1 June 2010 (wileyonlinelibrary.com) DOI 10.1002/sia.3408 MeV-energy probe SIMS imaging of major com- ponents in washed and fractured animal cells H. Yamada, a* Y. Nakata, a S. Ninomiya, b T. Seki, c T. Aoki, d J. Tamura e and J. Matsuo b Molecular images of pretreated animal cells were measured by secondary ion mass spectrometry (SIMS) with MeV-energy ion beam. SIMS analysis was performed for cell surfaces washed with buffers not only at high vacuum, but also at several tens Pa by using an orthogonal acceleration time of flight mass spectrometer (oaTOFMS) system. Moreover, molecular images of major components from washed and freeze fractured cells were obtained using MeV-energy probe SIMS. Copyright c  2010 John Wiley & Sons, Ltd. Keywords: SIMS; imaging mass spectrometry; swift heavy ions; animal cells Introduction Molecular imaging of biological samples with SIMS has attracted the attention of scientists because it offers the capability to uniquely identify chemical species with high sensitivity and high spatial resolution. For instance, SIMS imaging revealed the boron distribution in brain tumor cells at several tens of nm spatial reso- lution for a clinical boron neutron capture therapy (BNCT) agent. [1] Recently, demand for cellular-level imaging of drug metabolites, intact phospholipids or peptides has increased in the pharmaceu- tical and medical research fields. In general, SIMS imaging has been used for visualizing elemental distribution at the cellular level because of low molecular ion yields. To address this issue and open up a new possibility of SIMS imaging, we have developed a new system for imaging mass spectrometry (IMS) using MeV-energy heavy ion beams. Plasma desorption mass spectrometry (PDMS), which uses fission fragments from a 252 Cf source, has been used to measure biomolecules up to 20 kDa, such as antibiotics, peptides and proteins. [2,3] In a previous study, we successfully obtained the ion image of peptides up to 1 kDa by using a MeV-energy ion beam as a probe of SIMS measurements (MeV-SIMS) [4] Moreover, SIMS measurements at low vacuum (∼100 Pa) us- ing an orthogonal acceleration time of flight mass spectrometer (oaTOFMS) system and a continuous primary C 60 + beam were reported. [5,6] Molecular ions derived from fresh or undried biosam- ples could be directly detected using low vacuum or atmospheric SIMS systems. In this work, we demonstrate molecular imaging of animal cells with high sensitivity and spatial resolution. In addition, we applied MeV-energy ion beam to low-vacuum SIMS measurements and illustrated the capabilities of atmospheric SIMS and molecular imaging. Experimental Specimen Mouse fibroblast cells, 3T3-L1, and rat white lipid cells were purchased from DS Pharma Biomedical Co. (Osaka, Japan). The cells were seeded and cultured with Dulbecco’s Eagle’s mini- mum essential medium (MEM) (Nacalai Tesque, Kyoto, Japan), fatal bovine serum (HyClone, Utah, USA), and gentamicin (Nacalai Tesque) on 10 mm × 10 mm Si wafers. The Si wafers were steril- ized by flaming prior to cell seeding. After the cells were seeded and cultured overnight, their membranes and nuclear envelopes were stained with 3,3-dihexyloxacarbocyanine iodide (DiOC 6 (3)) (Nacalai Tesque). After staining, the cells were washed with phosphate-buffered saline (PBS) (Nacalai Tesque) and 0.1 mol/l ammonium acetate solution (Nacalai Tesque). Cryogenic sample preparation by the simple sandwich freeze- fracture method [1] exposes cell interiors for SIMS analysis. The animal cells on the substrate were sandwiched by another clean Si wafer and the sandwich was frozen in liquid nitrogen. The sand- wich was separated under liquid nitrogen. We used the fractured cells adhering on the top Si wafer to avoid signals derived from the culture medium for this analysis. The washed or fractured cells were then freeze-dried at −60 ◦ C for about 2 h onto a Peltier cooling unit, 9530/228/060M (Ferrotec Co., Tokyo, Japan). The Peltier unit was inserted into a vacuum chamber, at low pressure (10 −2 Pa). The dried samples were stored in a desiccator until SIMS measurements were performed. ∗ Correspondence to: H. Yamada, Department of Nuclear Engineering, Kyoto University, Sakyo, Kyoto, 606-8501, Japan . E-mail: hideaki-yamada@nucleng.kyoto-u.ac.jp a Department of Nuclear Engineering, Kyoto University, Sakyo, Kyoto, 606-8501, Japan b QuantumScienceandEngineeringCenter,KyotoUniversity,Uji,Kyoto611-0011, Japan c CREST, Japan Science and Technology Agency, Chiyoda, Tokyo, 102-0075, Japan d Department of Electronic Science and Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan e Mass Spectrometry Business Unit, JEOL Ltd., Akishima, Tokyo 196-8558, Japan Surf. Interface Anal. 2011, 43, 363–366 Copyright c  2010 John Wiley & Sons, Ltd.