Objective comparison of scanning ion and scanning electron microscope images

Common and different aspects of scanning electron microscope (SEM) and scanning ion microscope (SIM) images are discussed from a viewpoint of interaction between ion or electron beams and specimens. The SIM images [mostly using 30 keV Ga focused ion beam (FIB)] are sensitive to the sample surface as well as to low-voltage SEM images. Reasons for the SIM images as follows: (1) no backscattered-electron excitation; (2) low yields of backscattered ions; and (3) short ion ranges of 20–40nm, being of the same order of escape depth of secondary electrons (SE) [=(3–5) times the SE mean free path]. Beam charging, channeling, contamination, and surface sputtering are also commented upon.     

Focused ion beam/scanning electron microscopy studies of Porcellio scaber (Isopoda, Crustacea) digestive gland epithelium cells

The focused ion beam (FIB) was used to prepare cross sections of precisely selected regions of the digestive gland epithelium of a terrestrial isopod P. scaber (Isopoda, Crustacea) for scanning electron microscopy (SEM). The FIB/SEM system allows ad libitum selection of a region for gross morphologic to ultrastructural investigation, as the repetition of FIB/SEM operations is unrestricted. The milling parameters used in our work proved to be satisfactory to produce serial two-dimensional (2-D) cuts and/or three-dimensional (3-D) shapes on a submicrometer scale. A final, cleaning mill at lower ion currents was employed to minimize the milling artifacts. After cleaning, the milled surface was free of filament- and ridge-like milling artifacts. No other effects of the cleaning mill were observed.     

Comparison in spatial spreads of secondary electron information between scanning ion and scanning electron microscopy

Monte Carlo simulations have been carried out to compare the spatial spreads of secondary electron (SE) information in scanning ion microscopy (SIM) with scanning electron microscopy (SEM). Under Ga ion impacts, the SEs are excited by three kinds of collision-partners, that is, projectile ion, recoiled target atom, and target electron. The latter two partners dominantly contribute to the total SE yield gamma for the materials of low atomic number Z2. For the materials of high Z2, on the other hand, the projectile ions dominantly contribute to gamma. These Z2 dependencies generally cause the gamma yield to decrease with an increasing Z2, in contrast with the SE yield delta under electron impacts. Most of the SEs are produced in the surface layer of about 5lambda in depth (lambda: the mean free path of SEs), as they are independent of the incident probe. Under 30 keV Ga ion impacts, the spatial spread of SE information is roughly as small as 10 nm, decreasing with an increasing Z2. Under 10 keV electron impacts, the SEI excited by the primary electrons has a small spatial spread of about 5lambda, but the SEII excited by the backscattered electrons has a large one of several 10 to several 100 nanometers, decreasing with an increasing Z2. The main cause of a small spread of SE information at ion impact is the short ranges of the projectile ions returning to the surface to escape as backscattered ions, the recoiled target atoms, and the target electrons in collision cascade. The 30 keV Ga-SIM imaging is better than the 10 keV SEM imaging in spatial resolution for the structure/material measurements. Here, zero-size probes are assumed.     

Focused ion beam manipulation and ultramicroscopy of unprepared cells

The focused ion beam (FIB) technique of nanomachining combined with simultaneous scanning electron microscopy (SEM) was used for submicron manipulation and imaging of unprepared (fresh) cells to demonstrate the potentiality of the FIB/SEM technique for ultramicroscopic studies. Sectioning at the nanoscale level was successfully performed by means of ion beam-driven milling operations that reveal the ultrastructure of fresh yeast cells. The FIB/SEM has many advantages over other ultramicroscopy techniques already applied for unprepared/fresh biological samples.     

Controlling FIB‐SBEM slice thickness by monitoring the transmitted ion beam

Serial block‐face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack‐acquisition time to several days. Here, we describe a method for controlling the ion‐beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted‐beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion‐mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in‐column detector is explored.     

Post‐thinning using Ar ion‐milling system for transmission electron microscopy specimens prepared by focused ion beam system

We investigate Ar ion‐milling rates and Ga‐ion induced damage on sample surfaces of Si and GaAs single crystals prepared by focused ion beam (FIB) method for transmission electron microscopy observation. The convergent beam electron diffraction technique with Bloch simulation is used to measure the thickness of the Ar‐ion milled samples to calculate the milling rates of Si and GaAs single crystals. The measurement shows that an amorphous layer is formed on the sample surface and can be removed by further Ar‐ion milling. In addition, the local symmetry breaking induced by FIB is investigated using quantitative symmetry measurement. The FIBed‐GaAs sample shows local symmetry breaking after FIB milling, although the FIBed‐Si sample has no considerable symmetry breaking.     

Carbon fiber composite targets for nuclear fusion technology: a focused ion beam/scanning electron microscope investigation

Carbon fiber composite (CFC) targets are investigated by a focused ion beam/scanning electron microscope (FIB/SEM) in a joint project aiming at the development of robust divertors in the International Thermonuclear Experimental Reactor (ITER). These mockups are exposed to a plasma that simulates the off-normal thermal loads foreseen for ITER and display a rich, puzzling impact scenario. Morphological elements are identified at the exposed surface and beneath it, and are examined in order to point out the relevant processes involved. Each technique adopted is discussed and evaluated.     

Observation of human dentine by focused ion beam and energy-filtering transmission electron microscopy

Molar dentine was sliced into 100 nm ultrathin sections, by means of a focused ion beam, for observation by energy-filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10 nm wide and 50–100 nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.     

Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.     

Transmission electron microscopy of fluorapatite–gelatine composite particles prepared using focused ion beam milling

In this paper, synthetic fluorapatite–gelatine composite particles are prepared for transmission electron microscopy (TEM) studies using two methods based on focused ion beam (FIB) milling. TEM studies on the FIB‐prepared specimens are compared with TEM observations on samples prepared using an ultramicrotome. The results show that ultramicrotome slicing causes significant cracking of the apatite, whereas the ion beam can be used to make high‐quality, crack‐free specimens with no apparent ion beam‐induced damage. The TEM observations on the FIB‐prepared samples confirm that the fluorapatite composite particles are composed of elongated, preferentially orientated grains and reveal that the grain boundaries contain many small interstices filled with an amorphous phase.     

New method for characterizing paper coating structures using argon ion beam milling and field emission scanning electron microscopy

We have developed a new method for characterizing microstructures of paper coating using argon ion beam milling technique and field emission scanning electron microscopy. The combination of these two techniques produces extremely high-quality images with very few artefacts, which are particularly suited for quantitative analyses of coating structures. A new evaluation method has been developed by using marker-controlled watershed segmentation technique of the secondary electron images. The high-quality secondary electron images with well-defined pores makes it possible to use this semi-automatic segmentation method. One advantage of using secondary electron images instead of backscattered electron images is being able to avoid possible overestimation of the porosity because of the signal depth. A comparison was made between the new method and the conventional method using greyscale histogram thresholding of backscattered electron images. The results showed that the conventional method overestimated the pore area by 20% and detected around 5% more pores than the new method. As examples of the application of the new method, we have investigated the distributions of coating binders, and the relationship between local coating porosity and base sheet structures. The technique revealed, for the first time with direct evidence, the long-suspected coating non-uniformity, i.e. binder migration, and the correlation between coating porosity versus base sheet mass density, in a straightforward way.     

Development of environmental scanning electron microscopy electron beam profile imaging with self-assembled monolayers and secondary ion mass spectroscopy

A method for demonstrating the scattering of the primary electron beam in the presence of a gas has been developed. A self-assembled decanethiol monolayer is damaged by primary beam electrons. The damaged portion of the mono-layer is exchanged with another thiol-containing molecule by immersion in solution. The resulting film is imaged using a secondary ion mass spectrometer. Three-dimensional reconstruction of the data yields a representation of scattered electrons in the gaseous environment of the environmental scanning electron microscope.     

Localized grounding, excavation, and dissection using in-situ probe techniques for focused ion beam and scanning electron microscopy: experiments with rock varnish

While investigating rock varnish, we explored novel uses for an in‐situ micromanipulator, including charge collection, sample manipulation, as well as digging and dissection at the micron level. Dual‐beam focused ion beam microscopes (DB‐FIB or FIBSEM) equipped with micromanipulators have proven to be valuable tools for material science, semiconductor research, and product failure analysis. Researchers in many other disciplines utilize the DB‐FIB and micromanipulator for site‐specific transmission electron microscope (TEM) foil preparation. We have demonstrated additional applications for in‐situ micromanipulators. SCANNING 34: 279–283, 2012. © 2012 Wiley Periodicals, Inc.     

An experimental model of beam broadening in the variable pressure scanning electron microscope

In the variable pressure scanning electron microscope (VP-SEM) the incident electrons pass through a gaseous environment and the beam is scattered by these interactions. We show here that the experimental intensity profile of the scattered beam can be described as Gaussian in form to a high level of accuracy. This provides a rapid means of accounting for the effects of beam scatter in imaging and microanalysis because the standard deviation of the Gaussian is a simple function of parameters such as working distance, beam energy, gas type and pressure.     

Subsurface nanoindentation deformation of Cu-Al multilayers mapped in 3D by focused ion beam microscopy

A new technique for the three-dimensional analysis of subsurface damage of nanocomposites is presented. Cu–Al multilayers, grown epitaxially on (0001)Al₂O₃ single crystals by ultra high vacuum molecular beam epitaxy, have been deformed by nanoindentation. Systematic slicing and imaging of the deformed region by focused ion beam microscopy enables a 3D data set of the damaged region to be collected. From this 3D data set, profiles of the deformed sub-surface interfaces can be extracted. This enables the deformation of the individual layers, substrate and overall film thickness to be determined around the damage site. These 3D deformation maps have exciting implications for the analysis of mechanical deformation of nanocomposites on a sub-micrometre scale.     

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.     

Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

Low-temperature scanning electron microscopy in biology

A review of low-temperature scanning electron microscopy (LTSEM) with regard to preparation protocols, specimen preservation, experimental approaches, and high-resolution studies, is provided. Preparative procedures are described and recent developments in methodologies highlighted. It is now well established that LTSEM, for most biological specimens, provides superior specimen preservation than does ambient-temperature SEM. This is because frozen-hydrated samples retain most or all of their water, are rapidly immobilized and stabilized by cryofixation, and are not exposed to chemical modification or solvent extraction. Nevertheless, artefacts in LTSEM are common and most arise because frozen-hydrated specimens contain water. LTSEM can be used as a powerful experimental tool. Advantages of employing LTSEM for this purpose and ways in which it can be used for novel experimentation are discussed. The most exciting development in recent years has been high-resolution LTSEM. The advantages, problems and requirements for this approach are defined.     

Imaging of human colon cancer cells using He-Ion scanning microscopy

Due to its low beam current and charge compensation mechanism He-Ion scanning microscopy is a very promising tool for imaging biological cells. However, to obtain relevant information, the method used for sample preparation is also critical. In this work, we have used a Carl Zeiss Orion Plus helium-ion microscope to study the effect of sample gold coating on the morphology of human colorectal adenocarcinoma Caco2 cells. The fixative glutaraldehyde was used and the selective gold coating of the samples was investigated. A comparative study with standard scanning electron microscopy is presented.     

Experimental data and model simulations of beam spread in the environmental scanning electron microscope

This work describes the comparison of experimental measurements of electron beam spread in the environmental scanning electron microscope with model predictions. Beam spreading is the result of primary electrons being scattered out of the focused beam by interaction with gas molecules in the low-vacuum specimen chamber. The scattered electrons form a skirt of electrons around the central probe. The intensity of the skirt depends on gas pressure in the chamber, beam-gas path length, beam energy, and gas composition. A model has been independently developed that, under a given set of conditions, predicts the radial intensity distribution of the scattered electrons. Experimental measurements of the intensity of the beam skirt were made under controlled conditions for comparison with model predictions of beam skirting. The model predicts the trends observed in the experimentally determined scattering intensities; however, there does appear to be a systematic deviation from the experimental measurements.