The Effect of Magnesium Ions on Chromosome Structure as Observed by Helium Ion Microscopy Astari Dwiranti, 1 Tohru Hamano, 1 Hideaki Takata, 1,5 Shoko Nagano, 2 Hongxuan Guo, 3 Keiko Onishi, 2 Toshiyuki Wako, 4 Susumu Uchiyama, 1 and Kiichi Fukui 1, * 1 Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan 2 Surface Characterization Group, Nano Characterization Unit, Advanced Key Technologies Division, National Institute for Materials Science, Sengen, Tsukuba, Ibaraki 305-0047, Japan 3 Global Research Center for Environment and Energy Based on Nanomaterials Science, National Institute for Materials Science, Sengen, Tsukuba, Ibaraki 305-0047, Japan 4 Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai, Tsukuba, Ibaraki 305-8602, Japan 5 Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan Abstract: One of the few conclusions known about chromosome structure is that Mg 2+ is required for the organization of chromosomes. Scanning electron microscopy is a powerful tool for studying chromosome morphology, but being nonconductive, chromosomes require metal/carbon coating that may conceal informa- tion about the detailed surface structure of the sample. Helium ion microscopy ~HIM!, which has recently been developed, does not require sample coating due to its charge compensation system. Here we investigated the structure of isolated human chromosomes under different Mg 2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg 2+ on chromosome structure. Chromatin fiber information was obtained more clearly with uncoated than coated chromosomes. Our results suggest that both overall features and detailed structure of chromatin are signifi- cantly affected by different Mg 2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg 2+ treatment, while 0 mM Mg 2+ resulted in a less compact and more fibrous structure 11 nm in diameter. We conclude that HIM is a powerful tool for investigating chromosomes and other biological samples without requiring metal/carbon coating. Key words: human chromosome, chromosome structure, helium ion microscopy ~HIM!, magnesium ion ~Mg 2+ !, osmium coating I NTRODUCTION Attempts to elucidate chromosome higher-order structure have remained elusive ~Fukui & Uchiyama, 2007; Fukui, 2009; Bian & Belmont, 2012!. The ionic environment affects chro- mosome structure because cations induce the compaction of polynucleosome chains, which become 30 nm diameter fi- bers in vitro, although the existence of 30 nm chromatin fi- bers in native chromosomes is still controversial ~Eltsov et al., 2008; Grigoryev & Woodcock, 2012; Nishino et al., 2012!. There is long-standing evidence that the integrity of condensed metaphase chromosomes depends on the bind- ing of divalent cations. Previous studies have shown that high Mg 2+ concentration ~2 mM! preserves the hetero- chromatin in a condensed state ~Cole, 1967; Adolph et al., 1986; Caravaca et al., 2005!. Current research in the life sciences often requires ultra-high resolution microscopy techniques to provide de- tailed investigation of nanoscale structures, such as chroma- tin fibers at high resolution. Scanning electron microscopy ~SEM! has been useful in studying chromosome structure because it has higher resolution and magnification than optical microscopy. However, it requires laborious prepara- tion steps, including metal/carbon coating, since chromo- somes, like most biological samples, are nonconducting. Furthermore, metal/carbon coating may ultimately lead to misinterpretation of the data obtained; for example, the metal coating can form a deposit on the top of the sample, rendering a false surface structure ~Pretorius, 2010!. To prevent the accumulation of negatively charged electrons, an ionic liquid method has been applied ~Dwiranti et al., 2012!. However, important features can still be hidden. The development of a technique that enables biological samples to be observed without coating would greatly aid the study of biological structures, such as chromosomes. Helium ion microscopy ~HIM! has great potential for life science applications ~Morgan et al., 2006!. HIM resem- bles SEM but uses positively charged helium ions instead of negatively charged electrons to image the sample in the chamber. The application of HIM to biological samples has recently been demonstrated by the observation of human colorectal adenocarcinoma morphology Caco2 cells ~Bazou et al., 2011! and the epithelium of rat kidney ~Rice et al., 2013!. Received August 6, 2013; accepted October 4, 2013 *Corresponding author. E-mail: kfukui@bio.eng.osaka-u.ac.jp Microsc. Microanal. Page 1 of 5 doi:10.1017/S1431927613013792 Microscopy AND Microanalysis © MICROSCOPY SOCIETY OF AMERICA 2013