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.     

Scanning electron microscopic technique for imaging a diamond tool edge

A technique is described to measure the edge radius of diamond cutting tools using the scanning electron microscope (SEM). This method attempts to overcome two major limitations of the SEM in this application: low image contrast and lack of quantitative topographic information. A line of electron beam contamination, viewed at an angle, provides improved contrast for focusing and a means of obtaining the tool profile from the geometry.     

The method of increasing COMPO contrast by linearization of backscattering characteristic η =f(Z)

The backscattered electron signal (BSE) in the scanning electron microscope (SEM) has been used for investigation of a specimen surface composition (COMPO mode). Creation of a material composition map is difficult because the dependence of backscattering coefficient η on the atomic number Z for Z > 40 is nonlinear. The method of increase in SEM resolution for the BSE signal by use of digital image processing has been proposed. This method is called the linearization of the η =f(Z) characteristic. The function approximating the experimental η =f (Z) dependence was determined by numerical methods. After characteristics linearization, the digital image in COMPO mode allows to distinguish between two elements with high atomic numbers if their atomic numbers differ by ΔZ = 1.     

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.     

The reduction of the beam gas interactions in the variable pressure scanning electron microscope with the use of helium gas

This paper presents experimental observations on electron scattering by gases (helium and air) in the specimen chamber of a variable pressure scanning electron microscope. It shows an important reduction of the beam scattering with the use of helium gas, and the consequences for the x-ray microanalysis are discussed.     

S—250MK3型扫描电子显微镜故障及维修

介绍了剑桥Ｓ－２５０Ｍ型扫描电子显微镜在实际操作中出现的典型故障及维修方法。     

Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

Cryo-techniques applied to stratum corneum with description of a new sample holder for cryo-scanning electron microscopy of freeze-fractured samples

Stratum corneum structure greatly differs from that of the living epidermis and specific sample cryo-preparation techniques have to be used. Practical aspects of these cryo-techniques applied to stratum corneum are discussed. Emphasis is placed on scanning electron microscopy of cryo-fixed samples. A new sample holder designed for cryo-scanning electron microscopy of freeze-fractured stratum corneum is described.