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.     

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 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.     

Making EBSD on water ice routine

Electron backscatter diffraction (EBSD) on ice is a decade old. We have built upon previous work to select and develop methods of sample preparation and analysis that give >90% success rate in obtaining high‐quality EBSD maps, for the whole surface area (potentially) of low porosity (<15%) water ice samples, including very fine‐grained (<10 μm) and very large (up to 70 mm by 30 mm) samples. We present and explain two new methods of removing frost and providing a damage‐free surface for EBSD: pressure cycle sublimation and ‘ironing’. In general, the pressure cycle sublimation method is preferred as it is easier, faster and does not generate significant artefacts. We measure the thermal effects of sample preparation, transfer and storage procedures and model the likelihood of these modifying sample microstructures. We show results from laboratory ice samples, with a wide range of microstructures, to illustrate effectiveness and limitations of EBSD on ice and its potential applications. The methods we present can be implemented, with a modest investment, on any scanning electron microscope system with EBSD, a cryostage and a variable pressure capability.     

Preparation of transmission electron microscopy cross-section specimens of crack tips using focused ion beam milling

The preparation of transmission electron microscope (TEM) thin foil specimens from metal alloys containing cracks is usually thwarted by the difficulty in preventing preferential erosion of material along the flanks and at the tips of cracks. Recent developments in focused ion beam (FIB) micromachining methods have the potential to overcome this inherent problem. In this article we describe the development of new procedures, one using FIB alone and the other using a combination of FIB with more conventional ion milling to generate TEM specimens that largely retain the microstructural information at stress corrosion cracks in austentic alloys. Examples of corrosion product phase identification and interfacial segregation are included to verify that detailed information is not destroyed by ion bombardment during specimen preparation.     

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.     

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.     

Conventional and back-side focused ion beam milling for off-axis electron holography of electrostatic potentials in transistors

Off-axis electron holography is used to characterize a linear array of transistors, which was prepared for examination in cross-sectional geometry in the transmission electron microscope (TEM) using focused ion beam (FIB) milling from the substrate side of the semiconductor device. The measured electrostatic potential is compared with results obtained from TEM specimens prepared using the more conventional 'trench' FIB geometry. The use of carbon coating to remove specimen charging effects, which result in electrostatic fringing fields outside 'trench' specimens, is demonstrated. Such fringing fields are not observed after milling from the substrate side of the device. Analysis of the measured holographic phase images suggests that the electrically inactive layer on the surface of each FIB-milled specimen typically has a thickness of 100 nm.     

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.     

Preparation of site-specific cross-sections of heterogeneous catalysts prepared by focused ion beam milling

Focused ion beam (FIB) milling offers a novel approach to preparation of site‐specific cross‐sections of heterogeneous catalysts for examination in the transmission electron microscope (TEM). Electron‐transparent sections can be obtained without the need to embed or grind the original sample. Because the specimen can be imaged in the FIB with submicrometre resolution before, during and after milling it is possible to select precisely the region from which the section is removed and to control the thickness of the section to within tens of nanometres. The ability to produce sections in this way opens the possibility of studying a range of catalyst systems that have previously been impossible to examine with the TEM.     

Surface damage formation during ion-beam thinning of samples for transmission electron microscopy

All techniques employed in the preparation of samples for transmission electron microscopy (TEM) introduce or include artifacts that can degrade the images of the materials being studied. One significant cause of this image degradation is surface amorphization. The damaged top and bottom surface layers of TEM samples can obscure subtle detail, particularly at high magnification. Of the techniques typically used for TEM sample preparation of semiconducting materials, cleaving produces samples with the least surface amorphization, followed by low-angle ion milling, conventional ion milling, and focused ion beam (FIB) preparation. In this work, we present direct measurements of surface damage on silicon produced during TEM sample preparation utilizing these techniques. The thinnest damaged layer formed on a silicon surface was measured as 1.5 nm thick, while an optimized FIB sample preparation process results in the formation of a 22 nm thick damaged layer. Lattice images are obtainable from all samples.     

Combining Ar ion milling with FIB lift-out techniques to prepare high quality site-specific TEM samples

Focused ion beam (FIB) techniques can prepare site‐specific transmission electron microscopy (TEM) cross‐section samples very quickly but they suffer from beam damage by the high energy Ga⁺ ion beam. An amorphous layer about 20–30 nm thick on each side of the TEM lamella and the supporting carbon film makes FIB‐prepared samples inferior to the traditional Ar⁺ thinned samples for some investigations such as high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). We have developed techniques to combine broad argon ion milling with focused ion beam lift‐out methods to prepare high‐quality site‐specific TEM cross‐section samples. Site‐specific TEM cross‐sections were prepared by FIB and lifted out using a Narishige micromanipulator onto a half copper‐grid coated with carbon film. Pt deposition by FIB was used to bond the lamellae to the Cu grid, then the coating carbon film was removed and the sample on the bare Cu grid was polished by the usual broad beam Ar⁺ milling. By doing so, the thickness of the surface amorphous layers is reduced substantially and the sample quality for TEM observation is as good as the traditional Ar⁺ milled samples.     

Heating of TEM specimens during ion milling

Sample heating during preparation of electron-thin specimens for observation in transmission electron microscopy (TEM) can produce artefacts which invalidate observations. This is particularly true of two-phase materials such as metal matrix composites, for which sample cooling with liquid nitrogen cannot be used to preserve the substructure during milling. A series of experiments is conducted using an age-hardenable aluminium alloy which produces a trace of peak temperature attained by TEM specimens during ion milling. It is shown that peak temperatures of the order of 650 K can be attained using conventional milling parameters; the technique must therefore be used with caution with materials such as metal matrix composites. A simplified one-dimensional heat transfer analysis of the problem is conducted to show that the most critical parameter is heat transfer along the sample holder legs and across interfaces along the heat path. Temperature differentials within the TEM specimen are shown to be less significant, yet these alone are capable of reaching 60 K within a dimpled specimen under usual milling conditions.     

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.     

Off-axis electron holography of electrostatic potentials in unbiased and reverse biased focused ion beam milled semiconductor devices

Off‐axis electron holography in the transmission electron microscope (TEM) is used to measure two‐dimensional electrostatic potentials in both unbiased and reverse biased silicon specimens that each contain a single p–n junction. All the specimens are prepared for examination in the TEM using focused ion beam (FIB) milling. The in situ electrical biasing experiments make use of a novel specimen geometry, which is based on a combination of cleaving and FIB milling. The design and construction of an electrical biasing holder are described, and the effects of TEM specimen preparation on the electrostatic potential in the specimen, as well as on fringing fields beyond the specimen surface, are assessed.     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

Use of FIB to Study ZDDP Tribofilms

Focussed ion beam milling (FIB) followed by TEM has been used to study ZDDP tribofilms on rubbed steel surfaces. It has been found that the impact of high energy platinum and gallium ions during FIB causes significant morphological and structural changes to the uppermost 30–50 nm of a ZDDP tribofilm. This can be prevented by the low energy deposition of a quite thick gold layer prior to installation of the sample in the FIB facility. This problem, and its solution, have been quite widely reported in the non-tribology literature but have not previously been highlighted in the application of FIB to study tribological surfaces. It has also been found, using this gold pre-deposition method, that the bulk of the ZDDP tribofilm studied has a polycrystalline structure.     

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.     

The observation of intact hepatic endothelial cells by cryo-electron microscopy

Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum‐resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo‐electron microscopy (cryo‐EM) can be used as a good alternative for imaging samples as whole mounts. Cryo‐EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 °C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde‐fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae‐associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.     

Comparison of different preparation methods of biological samples for FIB milling and SEM investigation

When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.