Electron microscopy has been used to visualize chromosome since it has high resolution and magnification. However, biological samples need to be dehydrated and coated with metal or carbon before observation. Ionic liquid is a class of ionic solvent that possesses advantageous properties of current interest in a variety of interdisciplinary areas of science. By using ionic liquid, biological samples need not be dehydrated or metal‐coated, because ionic liquid behaves as the electronically conducting material for electron microscopy. The authors have investigated chromosome using ionic liquid in conjunction with electron microscopy and evaluated the factors that affect chromosome visualization. Experimental conditions used in the previous studies were further optimized. As a result, prewarmed, well‐mixed, and low concentration (0.5∼1.0%) ionic liquid provides well‐contrasted images, especially when the more hydrophilic and the higher purity ionic liquid is used. Image contrast and resolution are enhanced by the combination of ionic liquid and platinum blue staining, the use of an indium tin oxide membrane, osmium tetroxide‐coated coverslip, or aluminum foil as substrate, and the adjustment of electron acceleration voltage. The authors conclude that the ionic‐liquid method is useful for the visualization of chromosome by scanning electron microscopy without dehydration or metal coating. Microsc. Res. Tech. 75:1113–1118, 2012. © 2012 Wiley Periodicals, Inc.

One of the few conclusions known about chromosome structure is that Mg2+ 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 information 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 Mg2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg2+ 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 significantly affected by different Mg2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg2+ treatment, while 0 mM Mg2+ 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.

The structural details of chromosomes have been of interest to researchers for many years, but how the metaphase chromosome is constructed remains unsolved. Divalent cations have been suggested to be required for the organization of chromosomes. However, detailed information about the role of these cations in chromosome organization is still limited. In the current study, we investigated the effects of Ca2+ and Mg2+ depletion and the reversibility upon re-addition of one of the two ions. Human chromosomes were treated with different concentrations of Ca2+and Mg2+. Depletion of Ca2+ and both Ca2+ and Mg2+ were carried out using 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ethylenediaminetetraacetic acid (EDTA), respectively. Chromosome structure was examined by fluorescence microscopy and scanning electron microscopy. The results indicated that chromosome structures after treatment with a buffer without Mg2+, after Ca2+ depletion, as well as after depletion of both Mg2+, and Ca2+, yielded fewer compact structures with fibrous chromatin than those without cation depletion. Interestingly, the chromatin of EDTA-treated chromosomes reversed to their original granular diameters after re-addition of either Mg2+ or Ca2+ only. These findings signify the importance of divalent cations on the chromosome structure and suggest the interchangeable role of Ca2+ and Mg2+.

Abstract. Dewijanti ID, Mangunwardoyo W, Dwiranti A, Hanafi M, Artanti N. 2020. Short communication: Effects of the various source areas of Indonesian bay leaves (Syzygium polyanthum) on chemical content and antidiabetic activity. Biodiversitas 21: 1190-1195. Bay leaves (Syzygium polyanthum (Wight) Walp) are from an Indonesian plant species belonging to the Myrtaceae family. The Indonesian name for this plant is salam; it is found in Sumatra, Kalimantan, and the Java Islands. Its leaves are the part of the plant commonly used. Salam leaves are usually consumed by people as a food flavoring, but are also used as a traditional antidiabetic medicine. The purpose of this study was to discover the effects of salam leaves’ origins on their chemical content and antidiabetic activity. For this reason, salam leaf samples were collected from three different provinces in Java (West Java, Central Java, and East Java). The samples were extracted by boiling in water. Phytochemical screening, chemical analysis using TLC, HPLC, LC-MSMS, and FTIR, and antidiabetic in vitro testing using alpha-glucosidase inhibition methods were conducted on the salam leaves’ water extracts. The results showed that salam leaf extracts from all three provinces contained quercetin. However, the salam originating from East Java also contained coniferin, the salam from Central Java contained juncusol, and the salam from West Java contained retucine. The salam leaves originating from East Java showed the lowest antidiabetic activity, whereas salam leaf extract showed significantly higher antidiabetic activity. As conclusion, a plant’s growth origin affects its chemical content and antidiabetic activity.

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.