Chromosome observation by scanning electron microscopy using ionic liquid

Dwiranti, Astari; Lin, Linyen; Mochizuki, Eiko; Kuwabata, Susumu; Takaoka, Akio; Uchiyama, Susumu; Fukui, Kiichi

doi:10.1002/jemt.22038

Cited by 37 publications

(35 citation statements)

References 23 publications

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“…Because of these properties, ionic liquids have proven advantageous for biological sample preparation prior to SEM observation. In addition, platinum blue (Pt-blue) staining enhanced the contrast of chromosome images (Dwiranti et al, 2012). The ionic liquid technique provides visualization closer to the native state since it enables us to observe biological samples without drying, dehydration, or metal/carbon coating.…”

Section: Introductionmentioning

confidence: 99%

Chromosome Interior Observation by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Using Ionic Liquid Technique

et al. 2014

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Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.

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Section: Introductionmentioning

confidence: 99%

Chromosome Interior Observation by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Using Ionic Liquid Technique

et al. 2014

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“…Recently, Kuwabata et al (2006) showed that ILs could be observed by SEM without the electroncharge accumulation from the so-called charge-up effect, although ILs possess ionic conductivity. It was subsequently demonstrated that IL-treated specimens swollen with water could be observed by SEM without the laborious pretreatment processes (Arimoto et al, 2008c;Dwiranti et al, 2012;Ishigaki et al, 2011a, b, c;Takahashi et al, 2013;Tsuda et al, 2011b). These results led us to observe wet pollen grains treated with an IL under vacuum conditions.…”

Section: Introductionmentioning

confidence: 85%

SEM Observation of Wet Lily Pollen Grains Pretreated with Ionic Liquid

Komai

Okada

Inoue

et al. 2014

J. Japan. Soc. Hort. Sci.

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Mature pollen grains of Lilium cultivar, with their germ pores folded in upon themselves, were observed under a scanning electron microscope (SEM). The conventional pretreatment process requires aldehyde fixation, dehydration, drying and metal sputtering for SEM observation. These complicated and laborious procedures can considerably alter the morphology of pollen grains. In order to omit this conventional pretreatment process, we established a novel technique utilizing an ionic liquid (IL) that is composed solely of ions, namely, a liquid salt that can remain in a molten state even at room temperature. IL-treated pollen grains could be observed under vacuum conditions without artifacts, and furthermore, a satisfactory SEM image could visualize pollen grains in a wet state. The possible direction of future studies on ionic liquids in the SEM field is also discussed.

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“…For example, many types of ILs have been shown to exert antimicrobial activity. Another interesting application is to use ILs as a coating agent in scanning electron microscopy (SEM) of biological specimens . The nonvolatility of ILs makes it possible to observe SEM images of the specimen under wet conditions, and the high electronic conductivity of ILs prevents the specimen from charging.…”

Section: Introductionmentioning

confidence: 99%

Can cells maintain their bioactivity in ionic liquids? A novel single‐cell assessment by Raman microspectroscopy

Matsuda

Sakaguchi

Shigeto

2019

J Raman Spectroscopy

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Ionic liquids (ILs) are widely exploited in numerous applications in chemistry, biology, and materials science. Assessment of the toxicity of ILs is thus urgently needed to develop genuinely "green" ILs that do not pose hazards to the environment and humans. Despite considerable efforts mostly with cell viability assays, little has been understood about how, at the molecular level, ILs affect the bioactivity and intracellular chemistry of single individual cells. Here, we report a new method based on Raman microspectroscopy for quantitatively examining the effects of ILs on the bioactivity of single living fission yeast cells beyond just counting viable cells. The ILs studied are two hydrophilic ILs, 2-hydroxyethyl-trimethylammonium L-(+)-lactate [Cho][Lac] and 1-ethyl-3methylimidazolium acetate [emim][AcO], and they were mixed with liquid medium. We used a characteristic yeast Raman band at 1,602 cm −1 , which sharply reflects the bioactivity of yeast cells. For both ILs, the Raman spectra measured at lipid droplets in IL-treated yeast cells clearly exhibited the 1,602cm −1 band, indicating that yeast cells can indeed maintain their bioactivity in the ILs. Furthermore, we observed that the amide I peak in the cytoplasmic/nuclear Raman spectrum significantly blueshifts in [emim] [AcO] compared with that in the normal medium and [Cho][Lac]. This unexpected finding suggests an IL-induced change in protein secondary structure from αhelix to β-sheet and random-coil structures. The present study not only demonstrates that Raman microspectroscopy utilizing the 1,602-cm −1 band of yeast is a unique, powerful tool for single-cell assessment of IL toxicity, but it also calls attention to the effects of ILs on the structure and hence functions of intracellular molecules that might have been overlooked in previous studies.

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Chromosome observation by scanning electron microscopy using ionic liquid

Cited by 37 publications

References 23 publications

Chromosome Interior Observation by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Using Ionic Liquid Technique

Chromosome Interior Observation by Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Using Ionic Liquid Technique

SEM Observation of Wet Lily Pollen Grains Pretreated with Ionic Liquid

Can cells maintain their bioactivity in ionic liquids? A novel single‐cell assessment by Raman microspectroscopy

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