Beam transfer characteristics of a commercial environmental SEM and a low vacuum SEM

Two commercial instruments that permit a gaseous environment in their specimen chamber have been investigated, namely, a 'FEI Quanta 600 FEG' environmental scanning electron microscope and a 'LEO SUPRA 35VP FESEM' low vacuum scanning electron microscope. The gas flow field is first computed by the direct simulation Monte Carlo method and the gas density gradient, speed, Mach number and temperature are found in the transition region from high pressure to vacuum. The electron beam transfer characteristics are then derived for different accelerating voltages and pressures and a comparison is made among different situations and with some published works. Certain physical parameters are analysed and discussed to establish a figure of merit that can be used as a standard performance specification for commercial environmental scanning electron microscope and low vacuum scanning electron microscope.     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

Low pressure mode combined with OsO4 vapor fixation and sputter-coating for the preservation of delicate aerial hyphae and conidia in the ESEM

The newer generation of environmental scanning electron microscopes (ESEMs) allows samples to be viewed under a range of different vacuum conditions. No specific sample preparation protocols are required with the ESEMs, as fresh, unfixed samples are used and discarded later. We have worked out a method that preserves aerial hyphae on biltong that closely resemble fresh specimens and may be stored for viewing at a later date. Another advantage is that fixed samples are more resilient to the variable vacuum encountered in the ESEM. When biltong samples with fungal growth were first studied, we observed that vacuum-related artifacts were induced unless vacuum conditions and changes in pressure were carefully controlled. Damage readily occured in conidia and its delicate hyphae. Fresh, unfixed samples are very vulnerable to these artifacts. In addition, biltong proved to be a problematic study sample because of its high salt content, its hygroscopic nature as well as being laden with spices. To eliminate these artifacts, the preservation of specimens by OsO4 vapor fixation combined with a special sputter-coating technique is described. Previous studies confirmed that OsO4 vapor fixation is superior to traditional immersion fixation methods for the examination of hyphae and conidia of various fungi in a conventional SEM. However, both preparation methods induce sample shrinkage. We observed that OsO4 vapor fixation followed by Au coating under strictly controlled vacuum conditions induced fewer artifacts and gave the best images with minimum distortion in low pressure (LP) mode. The proposed method allows samples to be viewed both in ESEM and LP mode. There are however some disadvantages inherent to ESEM mode. Even when viewing fixed, coated samples, care should be taken to maintain a pressure of not lower than 0.2 Torr in the specimen chamber. It is critical that different samples have the same vacuum exposure history, as shrinkage and collapse were found to be directly related to the lowest pressure the samples were subjected to, both in the sputter coater chamber and in the electron microscope specimen chamber.     

真空吸具在球壳电子束焊接中的应用

分析球壳电子束焊接装夹的特点,运用真空吸具的优点,设计出一种快速、精确可靠的真空吸具辅助装夹系统,解决了长期以来球壳电子束焊接装夹找正难的问题。     

Optimum beam transfer in the environmental scanning electron microscope

The gas density of argon along the axis of a pressure-limiting aperture in an environmental scanning electron microscope is found by the direct simulation Monte Carlo method. The aperture is made on a thin material plate, producing the sharpest possible transition region between the specimen chamber and the differentially pumped region downstream of the gas flow. The entire regime from free molecule to continuum flow has been studied, which covers any size of aperture diameter and any pressure from vacuum to one atmosphere. The amount of electron beam transmitted without scattering at any point along the aperture axis is found in the range of accelerating voltage between 1 and 30 kV for argon. The electron beam transmission is further computed for helium, neon, hydrogen, oxygen, nitrogen and water vapour. This study constitutes the basis for the design and construction of an environmental scanning electron microscope having an optimum electron beam transfer, which is the primary requirement for an optimum performance instrument.     

Investigation of the morphology, viability and mechanical properties of yeast cells in environmental SEM

The mechanical properties of biological cells at nanoscale may be characterized using an environmental scanning electron microscopy (ESEM) combined with a force measurement device. However, the electron beam radiation in an ESEM may damage a specimen. So far, little is known about the radiation damage to biological cells. In this work, single yeast cells were imaged using an ESEM under both high and low vacuum modes. The changes in their morphology and viability were monitored as a function of radiation time for a given beam current of 538 pA corresponding to 10 kV accelerating voltage and spot size 4. Under the two modes, the radiation damage to the morphology of yeast cells became evident after an exposure time of 3 min, but under the low vacuum mode, the damage to their morphology was more severe. However, all cells lost their viability after 5 min under the high vacuum mode with the electron beam off from an initial viability of 95+/-1%. In contrast, the viability of cells under the low vacuum mode was found to be approximately 20% after 20 min. In addition, a newly developed ESEM-based nanomanipulation technique was applied to measure the force imposed on single yeast cells and their deformation, including contact diameter and central lateral diameter for the compression of single yeast cells to a given displacement within a time frame of 1 min, and the data obtained may be used to validate mathematical modeling of the stress-strain relationship for the compression of cells in order to determine their intrinsic mechanical property parameters.     

基于CFD技术管道气体流场计算与分析

基于CFD技术,运用CFD软件结合气体运动基本理论,以电子束焊接真空系统中管道设计为例,对中真空条件下气体在管道内的流动状态、速度、压力分布进行2D有限元数值模拟仿真和可视化研究,为真空系统管道参数确定和结构优化设计提供了有参考价值的数据。提出在真空系统设计选择前级泵时,为保证获得较大的抽速,达到高效与经济的效果,优先考虑主泵排气口直径与前级泵相匹配,再校核前级泵最大反压力和最大排气量,可以极大提高设计效率。     

Developing dual-beam methodologies for the study of heterogeneous polymer-based systems

The use of a combined focused ion beam/environmental scanning electron microscope (FIB/ESEM) offers new possibilities for imaging the internal structure of complex heterogeneous polymeric samples. The use of the focused ion beam, using positively charged gallium ions in conjunction with a measured 'defocused' low-energy primary electron beam, has permitted milling through the heterostructure to be achieved in a controlled way, exposing the inner structure, without introducing significant ion beam damage/destruction into the sample. The subsequent use of the environmental scanning electron microscope for imaging the revealed internal structure has then enabled insulating polymer structures to be imaged, without charging problems. Cross-sections of a 900-nm-thick spun cast film of phase-separated polystyrene–polybutadiene blends have been successfully milled and imaged; the morphology agreeing with previous results produced using ultramicrotomy and transmission electron microscopy.     

真空吸具在球壳电子束焊接中的应用

通过分析球壳电子束焊接装卡的特点，巧妙运用真空吸具的优点，设计出一种快速、精确可靠的真空吸具辅助装卡系统，解决了长期以来球壳电子束焊接装卡找正难的问题。     

Immunogold labelling in environmental scanning electron microscopy: applicative features for complementary cytological interpretation

We have combined environmental scanning electron microscopy (ESEM) and immunogold labelling (IGL) for the analysis of cell morphology and surface protein detection on human fine needle aspiration, which is processed in thin uniform monolayer (a single layer of cells) on a glass slide by Thin Prep technology. Among scanning electron microscopy techniques, we choose the environmental modality (ESEM) because it allows a slight manipulation of biological samples and an operational time comparable with cytological techniques. Moreover, the Thin Prep technology confirmed a reproducible cell monolayer on glass smear, minimizing problems for the determination of appropriate amount of material per slide. The first experimental data in ESEM-IGL on biological samples with fine needle aspiration Thin Prep, in human thyroid nodules, showed that cells retained their morphology and provided a clear IGL. The optimization of conditions (i.e. vacuum pressure, temperature and relative humidity) confirmed the possibility to observe an immunolabelled biological sample and morphological signal, joined with compositional informations, due to peculiar characteristics of gaseous secondary electron detector in ESEM. The ESEM-IGL and fine needle aspiration Thin Prep could be used in combination for the interpretation of cell morphology and cell surface immunolabelling. Our paper suggests this use as a powerful diagnostic tool in a pre-surgical evaluations, opening a new applicative window for electron microscopy.     

On the role of electron–ion recombination in low vacuum scanning electron microscopy

Here we demonstrate the effects of electron–ion recombination on imaging signals utilized in low vacuum scanning electron microscopes (SEMs). The presented results show that, under normal operating conditions, recombination of ionized gas molecules with secondary electrons (SEs) suppresses a significant fraction of emitted electrons. If the ion flux (and hence the spatial dependence of the SE–ion recombination rate) is laterally inhomogeneous across the imaged region of a specimen, contrast in SE images can be influenced and in some cases (under conditions of high detector field strength and long ionic mean free path) dominated by variations in the recombination rate. Consequently, SE images of features such as topographic asperities can exhibit edge‐darkening, leading to inversion of some topographic contrast. Recognition of the extent and nature of electron–ion recombination is required for a correct understanding of processes occurring in variable pressure SEMs and, subsequently, for models of image formation.     

A method to measure the total scattering cross section and effective beam gas path length in a low‐vacuum SEM

A method is presented to determine the total scattering cross section of imaging gases used in low‐vacuum scanning electron microscopy or environmental scanning electron microscopy. Experimental results are presented for water vapor, nitrogen gas and ambient air for primary beam electron energies between 5 and 30 keV. The measured results are compared and discussed with calculated values. This method allows the effective beam gas path length (BGPL) to be determined. The variations of the effective BGPL with varying chamber pressure are presented. SCANNING 31: 107–113, 2009. © 2009 Wiley Periodicals, Inc.     

Infrared thermometers for small wires drawing

This work describes the design of two contactless thermometers based respectively on a total radiance and two-color pyrometry scheme, developed to measure the temperature of a small brass coated steel wire during wire drawing. In this critical condition, wire oscillation and relevant insertion errors do not allow using commercial contact or contactless sensors. Thus, ad hoc pyrometers optical layouts have been designed in order to minimize sensitivity to the wire oscillations and emissivity changes. Moreover, performances associated to different infrared detectors have been compared using as figure of merit the achieved measurement uncertainty simulating typical disturbances, i.e. the background temperature variation, the slope of the wire’s emissivity and the effect of the atmosphere transmittance. Finally, the uncertainty budgets were drawn, evidencing the limitations of the proposed methods and identifying the best configuration for both developed instruments.     

Optimization of a Wolter type I mirror for a soft X-ray microscope

The demand for an X-ray microscope has received much attention because of the desire to study living cells under high resolution. A Wolter type I mirror used for soft X-ray microscope optics has a number of advantages. Although much progress has been made, it is still not easy to fabricate this mirror and satisfy the surface roughness and figure error requirements. From the mirror fabrication point of view, it is necessary to see the mirror design and the tolerance budget, especially with respect to the surface roughness and the figure errors. This paper deals with the design and optimization of a Wolter type I microscope mirror. The optimization was carried out by choosing an optimum central grazing incidence angle for which a merit function had the maximum value. The image quality of the mirror was also examined. A smaller diameter gave better image quality because of the Abbe sine condition. Finally, the figure errors for the axial and the radial directions were simulated by sinusoidal deformation waves, and the figure tolerance was obtained.     

Charge and charging compensation on oxides and hydroxides in oxygen environmental SEM

Oxygen environment was applied to the scanning electron microscopy (SEM) analysis of insulating samples. In the high vacuum SEM, a local oxygen pressure was provided, and in the environmental SEM, oxygen atmosphere was used instead of water, the commercial mode. The charging effects in the SEM observation and component characterization of samples such as Al(2)O(3), Al(OH)(3), Mg(OH)(2) and others can be eliminated or significantly reduced. The oxygen environment does not only provide a new approach to releasing the charging difficulty in the analyses using electron beam as a probe, but also provide an insightful hint to the understanding of the charging processes in general.     

Design and construction of an atmospheric or environmental SEM (part 1)

Progress in designing and constructing an atmospheric or environmental scanning electron microscope (SEM) is reported. The introduction of vacuum pumping between the objective and pressure limiting aperture has allowed the use of relatively large pressure limiting apertures, i.e., up to 57 μm for operation at atmospheric pressure or up to 400 μm for operation at saturation water vapour pressure and room temperature. The imaging obtained has been considerably improved by these developments. The first part of experimentation and analysis on the vacuum characteristics of the new system together with different detection configurations is also presented.     

The structure of grease via electron microscopy and image analysis

Many of the insolubles that are essential to the structure of lubricating greases are too small to be usefully imaged in the optical microscope. The higher resolution of the electron microscope is necessary to examine the thickener and additive morphology that contribute so much to the performance of modern greases. However, the high vacuum requirements of these instruments mean that the oil component of grease should not be introduced into the microscope at ambient temperatures. Techniques have been developed to remove the oil prior to examination, or to reduce its vapour pressure to a negligible level. A number of such techniques which have proved to be of value for both the scanning (SEM) and transmission (TEM) microscopes are described and illustrated. The distribution of the thickener fibres can now be measured and analysed by an image analysis procedure from the images of freeze-fracture TEM replicas.     

Consequences of positive ions upon imaging in low vacuum scanning electron microscopy

The effects caused by an excess quantity of ionized gas molecules within the low vacuum, variable pressure and environmental scanning electron microscope (ESEM) are described with reference to mechanisms by which they can influence imaging conditions. These effects can include specimen charging, recombination and development of space charge. They are demonstrated for three different classes of sample: (1) an electrically grounded conductor, (2) an electrically floating conductor, and (3) an electrical insulator. A new device is presented that will aid excess charge removal within the ESEM and help correct for some of these effects, thereby dramatically improving imaging over a wide range of operating conditions and samples. The mechanism of image enhancement is demonstrated with reference to the three classes of sample described above.     

高分辨二次离子质谱超高真空样品台的研制

目前,微束类分析仪器的一次离子束斑越来越精细,直径已达到微米级甚至亚微米级,仪器真空度也已达到稳定的超高真空水平,因此,亟需研制一种与之相匹配的高分辨样品台,提高在超高真空环境下的分辨力及定位精度。新研制的样品台,通过置于真空腔体外部的高精度滑台驱动小型焊接波纹管的伸缩传递三维运动,解决了大尺寸焊接波纹管表面放气及承受较大大气压力的问题;通过使用适用于超高真空的材料和固体润滑、无动态密封,解决了高真空中摩擦偶件的润滑问题。实验数据表明：在40 mm行程范围内,运动平台三轴定位重复性为0.94、1.83、0.38 μm,分辨力为0.10 μm,并已成功地应用在1.19×10^-6 Pa超真空环境下。外置的高精度滑台不仅克服了真空电机在真空腔体内发热及绝缘材料放气的问题,而且不需要引入任何电子电气元件及导线,不会在分析腔室内产生电磁干扰,行程不受装置本身的限制。这些特性不仅可满足二次离子质谱仪的要求,也适用于具有高分辨、超高真空环境、无污染要求的其他分析测试仪器。     

Skirting: a limitation for the performance of X-ray microanalysis in the variable pressure or environmental scanning electron microscope

The variable pressure or environmental scanning electron microscope (VP-SEM; ESEM) has become the microscope of choice for many scientists and technologists. Hence, the development of robust methods for X-ray microanalysis, limited by skirting, has become critical. In this paper, two pressure variation correction methods (Doehne and Gauvin) are compared. Both of these methods appear to be effective; the results were found to be well within 10% of the values obtained at 0 Pa. The Doehne method is dependent on an empirical factor (D), therefore the accuracy of the results will depend on the accuracy of this value. Also the Doehne method is compromised by the nonlinearity of the response with pressure. The Gauvin method is more user-friendly and more precise when considering the total range of pressure.