Supramolecular structure of humic acids by TEM with improved sample preparation and staining

Understanding the role of the dissolved organic matter in the environment will benefit from the characterization of its components at the supramolecular scale. With its very high spatial resolution the transmission electron microscope (TEM) allows the determination of colloidal size, structure, and shape on aggregated substances as well as single particles. It also allows the determination of the chemical composition if coupled with an X-ray detector. However, TEM preparation artifacts may hamper the relevance of such observations. This study demonstrates the capacity of TEM for the size and shape analysis of humic substances. Three sample preparation techniques (ultramicrotomy sectioning after embedding into a hydrophilic resin, drop deposition, and absorption) were evaluated with the Fluka humic acid (HA). Additionally, several staining agents (cesium hydroxide, lead citrate, uranyl acetate, and ruthenium tetraoxide) were used to increase the humic acid contrast for TEM observations. An improved simple preparation method with selective staining of the HA is proposed which permits the imaging of the humic acid macromolecules. The results show that the Fluka humic acid is composed mainly of fibrils generated by the interconnection of basic molecules of about 20 nm diameter with some isolated larger macromolecules (30-200 nm).     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Imaging and analysis of Bacillus anthracis spore germination

External and internal changes occurring during the process of germination of Bacillus anthracis spores were observed through atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. AFM studies showed that in response to L-alanine (4 mM), as a germinant, the spore germinates into a vegetative cell in 3 hours. The temporal size changes occurring during the germination were gradual but the major change in size was observed between the second and third hour. TEM of spores showed the presence of varied layers, which is in accordance with previous studies. However, the integrity of these layers was lost gradually during the process of germination. The inner spore membrane remains intact even until late stages of germination, whereas the coat, outer spore membrane, and the cortical layers are discarded at the second-hour stage. The results indicate that sequential changes during the germination of a B. anthracis spore are similar to other species of the Bacillus group.     

Transmission electron microscopic observations of membrane effects of antibiotic cecropin B on Escherichia coli

The pathway of cell membrane lysis by the peptide antibiotic cecropin B (CB), which contains both a hydrophobic and an amphipathic alpha-helix, was analysed by assessing the morphological changes of Escherichia coli following treatment with the peptide. Exposure of green fluorescent protein (GFP)-expressing E. coli to CB does not lead to an efflux of GFP. Moreover, transmission electron microscopic (TEM) examination of cecropin B-treated cells showed that severe swelling precedes cell death and that the outer membrane becomes distended away from the plasma membrane. Using immuno-gold staining and TEM of E. coli expressing the maltose-binding protein in the cytoplasm, it was apparent that the protein remains restricted to the cytoplasmic compartment. These observations suggest that CB causes gross disruption of the outer membrane of Gram-negative bacteria. Circular dichroism measurements of CB in the presence of cell membrane-mimicking liposomes showed that CB forms secondary structure dependent on the ratio of [lipid]/[peptide]. These observations from this study are important for the future design of custom antimicrobial peptides.     

Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating voltage of E = 1 keV and an ion-beam dose of It = 360 ～ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ～ 2 keV and It = 500 μA. min.     

An approach to the reduction of hydrocarbon contamination in the scanning electron microscope

The deposition of electron beam-induced specimen contamination in both the transmission (TEM) and scanning electron microscopes (SEM) has remained a problem since the beginning of these forms of microscopy. Generally, sources of SEM contamination can be attributed to one or a combination of three major contributors: (1) the pumping system; (2) outgassing of other internal SEM component parts (i.e., specimen stage, stage lubricants, O-rings, etc.), or (3) the sample (including its preparation and handling). Generally, because of the nature of SEM, specimen contamination can be minimized but is difficult to avoid fully. This work outlines three approaches taken with instruments at NIST to reduce the deposition of contamination in high-resolution cold-field emission SEMs. With some modification these techniques could be applied to any SEM. These approaches have been in successful operation for several years, resulting in a reduction in electron beam-induced hydrocarbon contamination.     

JEM-2100f透射电镜测角台的维护与故障处理

简述了JEM-2100f透射电镜测角台结构及样品杆进样、取样工作原理,分析了引起测角台故障的原因,阐述了测角台故障处理方法及日常维护技巧。     

W/B4C、W/C、W/Si多层膜的研究

给出了W/B₄C、W/C、W/Si(钨/碳化硼、钨/碳、钨/硅)周期多层膜的制备和测量研究。用超高真空直流磁控溅射方法制备出周期在1.1~7.2 nm范围内的多层膜样品,采用X射线衍射仪(XRD)小角度测量方法检测多层膜的光学性能,并用透射电镜(TEM)对样品的微观结构进行了研究。结果表明:周期大于1.3 nm的多层膜样品的结构质量高,膜层结构清晰,界面粗糙度小;周期为1.15 nm的多层膜的膜层结构不是很明显;所有膜层均为非晶态,没有晶相生成。结果还表明:采用目前的溅射设备和工艺过程能够制备出满足同步辐射荧光光束线上单色器用多层膜。     

Transmission electron microscopy study of crystallization in Fe-Si–B–Cr–C amorphous alloy

The crystallization behaviour of Fe₇₂Si_9.1B_14.8Cr_2.2C_1.9 alloy was studied. Three forms of the examined alloy were studied: water‐atomized powder, as‐spun ribbon and fully crystallized ribbon. Transmission electron microscopy studies of the examined alloy in the form of powder revealed partial crystallization of Fe₂B. The as‐spun ribbon was found to be fully amorphous, and no evidence of any crystalline phases was detected. Formation of metastable phases in the fully devitrificated ribbon was observed. The examined alloy, heated to above its crystallization temperature, consisted of α‐Fe₃(Si), Fe₂₃(C,B)₆, Fe₃B and Fe₂B crystalline phases.     

Electron beam modifications of InP surfaces

InP surfaces have been modified by using the intense electron beam of a V.G. HB501 STEM and subsequently examined in the same instrument using the reflection microscopy geometry. The surface changes can be divided into two parts; first, a fairly local removal of material, and second, more widespread effects including faceting and the formation of an oxide layer which extends for several microns around the point of incidence of the electron beam.