Image quality evaluation for FIB-SEM images

Focused ion beam scanning electron microscopy (FIB-SEM) tomography is a serial sectioning technique where an FIB mills off slices from the material sample that is being analysed. After every slicing, an SEM image is taken showing the newly exposed layer of the sample. By combining all slices in a stack, a 3D image of the material is generated. However, specific artefacts caused by the imaging technique distort the images, hampering the morphological analysis of the structure. Typical quality problems in microscopy imaging are noise and lack of contrast or focus. Moreover, specific artefacts are caused by the FIB milling, namely, curtaining and charging artefacts. We propose quality indices for the evaluation of the quality of FIB-SEM data sets. The indices are validated on real and experimental data of different structures and materials.     

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.     

Analysis of FIB‐induced damage by electron channelling contrast imaging in the SEM

We have investigated the Ga⁺ ion‐damage effect induced by focused ion beam (FIB) milling in a [001] single crystal of a 316 L stainless steel by the electron channelling contrast imaging (ECCI) technique. The influence of FIB milling on the characteristic electron channelling contrast of surface dislocations was analysed. The ECCI approach provides sound estimation of the damage depth produced by FIB milling. For comparison purposes, we have also studied the same milled surface by a conventional electron backscatter diffraction (EBSD) approach. We observe that the ECCI approach provides further insight into the Ga⁺ ion‐damage phenomenon than the EBSD technique by direct imaging of FIB artefacts in the scanning electron microscope. We envisage that the ECCI technique may be a convenient tool to optimize the FIB milling settings in applications where the surface crystal defect content is relevant.     

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.     

Focussed ion beam nanotomography reveals the 3D morphology of different solid phases in hardened cement pastes

Due to the development of integrated low-keV back-scattered electron detectors, it has become possible in focussed ion beam nanotomography to segment not only solid matter and porosity of hardened cement paste, but also to distinguish different phases within the solid matter. This paper illustrates a method that combines two different approaches for improving the contrast between different phases in the solid matrix of a cement paste. The first approach is based on the application of a specially developed 3D diffusion filter. The second approach is based on a modified data-acquisition procedure during focussed ion beam nanotomography. A pair of electron images is acquired for each slice in the focussed ion beam nanotomography dataset. The first image is captured immediately after ion beam milling; the second image is taken after a prolonged exposure to electron beam scanning. The acquisition of complementary focussed ion beam nanotomography datasets and processing the images with a 3D anisotropic diffusion filter allows distinguishing different phases within the hydration products.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

Effects of focused ion beam milling on electron backscatter diffraction patterns in strontium titanate and stabilized zirconia

This study investigates the effect of focused ion beam (FIB) current and accelerating voltage on electron backscatter diffraction pattern quality of yttria-stabilized zirconia (YSZ) and Nb-doped strontium titanate (STN) to optimize data quality and acquisition time for 3D-EBSD experiments by FIB serial sectioning. Band contrast and band slope were used to describe the pattern quality. The FIB probe currents investigated ranged from 100 to 5000 pA and the accelerating voltage was either 30 or 5 kV. The results show that 30 kV FIB milling induced a significant reduction of the pattern quality of STN samples compared to a mechanically polished surface but yielded a high pattern quality on YSZ. The difference between STN and YSZ pattern quality is thought to be caused by difference in the degree of ion damage as their backscatter coefficients and ion penetration depths are virtually identical. Reducing the FIB probe current from 5000 to 100 pA improved the pattern quality by 20% for STN but only showed a marginal improvement for YSZ. On STN, a conductive coating can help to improve the pattern quality and 5 kV polishing can lead to a 100% improvement of the pattern quality relatively to 30 kV FIB milling. For 3D-EBSD experiments of a material such as STN, it is recommended to combine a high kV FIB milling and low kV polishing for each slice in order to optimize the data quality and acquisition time.     

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.     

Surface damage induced by FIB milling and imaging of biological samples is controllable

Focused ion beam (FIB) techniques are among the most important tools for the nanostructuring of surfaces. We used the FIB/SEM (scanning electron microscope) for milling and imaging of digestive gland cells. The aim of our study was to document the interactions of FIB with the surface of the biological sample during FIB investigation, to identify the classes of artifacts, and to test procedures that could induce the quality of FIB milled sections by reducing the artifacts. The digestive gland cells were prepared for conventional SEM. During FIB/SEM operation we induced and enhanced artifacts. The results show that FIB operation on biological tissue affected the area of the sample where ion beam was rastering. We describe the FIB-induced surface major artifacts as a melting-like effect, sweating-like effect, morphological deformations, and gallium (Ga(+)) implantation. The FIB induced surface artifacts caused by incident Ga(+) ions were reduced by the application of a protective platinum strip on the surface exposed to the beam and by a suitable selection of operation protocol. We recommend the same sample preparation methods, FIB protocol for milling and imaging to be used also for other biological samples.     

Dopant profiling of focused ion beam milled semiconductors using off-axis electron holography; reducing artifacts,extending detection limits and reducing the effects of gallium implantation

Focused ion beam (FIB) milling is one of the few specimen preparation techniques that can be used to prepare parallel-sided specimens with nm-scale site specificity for examination using off-axis electron holography in the transmission electron microscope (TEM). However, FIB milling results in the implantation of Ga, the formation of amorphous surface layers and the introduction of defects deep into the specimens. Here we show that these effects can be reduced by lowering the operating voltage of the FIB and by annealing the specimens at low temperature. We also show that the electrically inactive thickness is dependent on both the operating voltage and type of ion used during FIB milling.     

Far‐reaching geometrical artefacts due to thermal decomposition of polymeric coatings around focused ion beam milled pigment particles

Focused ion beam and scanning electron microscope (FIB‐SEM) instruments are extensively used to characterize nanoscale composition of composite materials, however, their application to analysis of organic corrosion barrier coatings has been limited. The primary concern that arises with use of FIB to mill organic materials is the possibility of severe thermal damage that occurs in close proximity to the ion beam impact. Recent research has shown that such localized artefacts can be mitigated for a number of polymers through cryogenic cooling of the sample as well as low current milling and intelligent ion beam control. Here we report unexpected nonlocalized artefacts that occur during FIB milling of composite organic coatings with pigment particles. Specifically, we show that FIB milling of pigmented polysiloxane coating can lead to formation of multiple microscopic voids within the substrate as far as 5 μm away from the ion beam impact. We use further experimentation and modelling to show that void formation occurs via ion beam heating of the pigment particles that leads to decomposition and vaporization of the surrounding polysiloxane. We also identify FIB milling conditions that mitigate this issue.     

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.     

Focused ion beam milling for prototyping 2D and 3D photonic structures

Focused ion beam (FIB) milling is widely used in fields such as the semiconductor industry and materials science research. The direct writing and small fea     

Low damage lamella preparation of metallic materials by FIB processing with low acceleration voltage and a low incident angle Ar ion milling finish

Metallic materials are known to be very sensitive to Gallium (Ga) focused ion beam (FIB) processing. Crystal defects formed by FIB irradiation degrade the transmission electron microscope image quality, and it is difficult to distinguish original defects from FIB process-induced damage. A solution to this problem is the low acceleration voltage and low incident angle (LVLA) Argon ion milling, which can be incorporated as an extensional countermeasure for FIB damage removal and eventually for preparation of high-quality lamellae. The transmission electron microscope image quality of iron single crystal could be improved by removing crystal defects using the low acceleration voltage and low incident angle Argon ion milling finish. Lamella quality of the processing result was almost similar with that of the conventional electrolytic polishing. As a practical application of the process, low damage lamella of stainless cast steel could be prepared. Effectiveness of the FIB system equipped with the low acceleration voltage and low incident angle Argon ion milling function as a tool to make high-quality metallic material lamellae is illustrated.     

Focused ion beam milling of vitreous water: prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples

The feasibility of using a focused ion beam (FIB) for the purpose of thinning vitreously frozen biological specimens for transmission electron microscopy (TEM) was explored. A concern was whether heat transfer beyond the direct ion interaction layer might devitrify the ice. To test this possibility, we milled vitreously frozen water on a standard TEM grid with a 30‐keV Ga⁺ beam, and cryo‐transferred the grid to a TEM for examination. Following FIB milling of the vitreous ice from a thickness of approximately 1200 nm to 200–150 nm, changes characteristic of heat‐induced devitrification were not observed by TEM, in either images or diffraction patterns. Although numerous technical challenges remain, it is anticipated that ‘cryo‐FIB thinning’ of bulk frozen‐hydratred material will be capable of producing specimens for TEM cryo‐tomography with much greater efficiency than cryo‐ultramicrotomy, and without the specimen distortions and handling difficulties of the latter.     

Minimization of focused ion beam damage in nanostructured polymer thin films

Focused ion beam (FIB) instruments have proven to be an invaluable tool for transmission electron microscopy (TEM) sample preparation. FIBs enable relatively easy and site-specific cross-sectioning of different classes of materials. However, damage mechanisms due to ion bombardment and possible beam heating effects in materials limit the usefulness of FIBs. Materials with adequate heat conductivity do not suffer from beam heating during FIB preparation, and artifacts in materials such as metals and ceramics are primarily limited to defect generation and Ga implantation. However, in materials such as polymers or biological structures, where heat conductivity is low, beam heating can also be a problem. In order to examine FIB damage in polymers we have undertaken a systematic study by exposing sections of a PS-b-PMMA block copolymer to the ion beam at varying beam currents and sample temperatures. The sections were then examined by TEM and scanning electron microscopy (SEM) and analyzed using electron energy loss spectroscopy (EELS). Our empirical results show beam heating in polymers due to FIB preparation can be limited by maintaining a low beam current (≤100 pA) during milling.     

The evolution of indium precipitation in gallium focused ion beam prepared samples of InGaAs/InAlAs quantum wells under electron beam irradiation

Gallium ion (Ga⁺) beam damage induced indium (In) precipitation in indium gallium arsenide (InGaAs)/indium aluminium arsenide (InAlAs) multiple quantum wells and its corresponding evolution under electron beam irradiation was investigated by valence electron energy loss spectroscopy (VEELS) and high-angle annular dark-field imaging (HAADF) in scanning transmission electron microscopy (STEM). Compared with argon ion milling for sample preparation, the heavier projectiles of Ga⁺ ions pose a risk to trigger In formation in the form of tiny metallic In clusters. These are shown to be sensitive to electron irradiation and can increase in number and size under the electron beam, deteriorating the structure. Our finding reveals the potential risk of formation of In clusters during focused ion beam (FIB) preparation of InGaAs/InAlAs quantum well samples and their subsequent growth under STEM-HAADF imaging, where initially invisible In clusters of a few atoms can move and swell during electron beam exposure.     

Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling

Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Metrological challenges for reconstruction of 3‐D microstructures by focused ion beam tomography methods

The development of combined focused ion beam and scanning electron microscopes has enabled significant advances in the characterization of the 3‐D structure of materials. The repeated removal of thin layers or slices with an ion beam and imaging or mapping the chemical or crystallographic structure of each slice enables a 3‐D reconstruction from the images or maps. The accuracy of the reconstruction thus depends on the accuracy with which the slice thickness is measured and maintained throughout the process, and the alignment accuracy of the slices achieved during acquisition or by postacquisition corrections. A survey of papers published in this field suggests that the reconstruction accuracy is not often considered or reported. Using examples from examination of the 3‐D structure of hardmetals, issues affecting the accuracy of slice thicknesses and image realignments are examined and illustrated and potential errors quantified by the use of fiducial markers and the expected isotropy of the hardmetal structure itself.