NanoSIMS imaging of Bacillus spores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top‐sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift‐out. The lift‐out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top‐cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ～300 nm for top‐cut sections versus ～100 nm for electron‐transparent sections.     

Ion beam polishing for three‐dimensional electron backscattered diffraction

Serial sectioning by focused ion beam milling for three‐dimensional electron backscatter diffraction (3D‐EBSD) can create surface damage and amorphization in certain materials and consequently reduce the EBSD signal quality. Poor EBSD signal causes longer data acquisition time due to signal averaging and/or poor 3D‐EBSD data quality. In this work a low kV focused ion beam was successfully implemented to automatically polish surfaces during 3D‐EBSD of La‐ and Nb‐doped strontium titanate of volume 12.6 × 12.6 × 3.0 μm. The key to achieving this technique is the combination of a defocused low kV high current ion beam and line scan milling. The line scan was used to restrict polishing to the sample surface and the ion beam was defocused to ensure the beam contacted the complete sample surface. In this study 1 min polishing time per slice increases total acquisition time by approximately 3.3% of normal 3D‐EBSD mapping compared to a significant increase of indexing percentage and pattern quality. The polishing performance in this investigation is discussed, and two potential methods for further improvement are presented.     

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.     

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.     

On correction of translational misalignments between section planes in 3D EBSD

Three‐dimensional electron backscatter diffraction allows obtaining the 3D image of a material from the stack of 2D sections. This is achieved by repeated application of two different beams; electron beam for electron backscatter diffraction mapping of the surface and focused ion beam for removing a thin layer of material from the surface. In most of these systems with two beams, the experiment requires stage movements for correct positioning of the sample to the respective beams. However, imperfections in this positioning are difficult to avoid, which yield small translational misalignments between the sections in the output data. In this work, we deal with an important task of correcting these misalignments between the sections such that the 3D image is recovered properly. On a simple example, we demonstrate that commonly used methods fail in case there is a structural anisotropy in the material under consideration. We propose an improved alignment algorithm which can neglect this behaviour with the use of external support information on a systematic trend in the translational misalignments. Efficiency of the algorithm is proven on a number of simulated data with different kinds of anisotropy. Application to a real data sample of a fine grained aluminium alloy is also given. The algorithm is available in an open‐source library.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

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.     

Image contrast enhancement of Ni/YSZ anode during the slice‐and‐view process in FIB‐SEM

Focused ion beam‐scanning electron microscopy (FIB‐SEM) is a widely used and easily operational equipment for three‐dimensional reconstruction with flexible analysis volume. It has been using successfully and increasingly in the field of solid oxide fuel cell. However, the phase contrast of the SEM images is indistinct in many cases, which will bring difficulties to the image processing. Herein, the phase contrast of a conventional Ni/yttria stabilized zirconia anode is tuned in an FIB‐SEM with In‐Lens secondary electron (SE) and backscattered electron detectors. Two accessories, tungsten probe and carbon nozzle, are inserted during the observation. The former has no influence on the contrast. When the carbon nozzle is inserted, best and distinct contrast can be obtained by In‐Lens SE detector. This method is novel for contrast enhancement. Phase segmentation of the image can be automatically performed. The related mechanism for different images is discussed.     

3D correlative morphological and elemental characterization of materials at the deep submicrometre scale

This paper shows how X‐ray computed nanotomography (CNT) can be correlated with focused ion beam time‐of‐flight secondary ion mass spectrometry (FIB‐TOF‐SIMS) tomography on the same sample to investigate both the morphological and elemental structure. This methodology is applicable to relatively large specimens with dimensions of several tens of microns whilst maintaining a high spatial resolution of the order of 100 nm. However, combining X‐ray CNT and FIB‐TOF‐SIMS tomography requires innovative sample preparation protocols to allow both experiments to be conducted on exactly the same sample without chemically or structurally modifying the sample between measurements. Moreover, dedicated algorithms have been developed for effective data fusion that is biased with nine degrees of freedom. This methodology has been tested using a porous and heterogeneous solid oxide fuel cell (SOFC) that has features varying in size by three orders of magnitude – from hundreds of nanometre large pores and grains to tens of micron wide functional layers.     

Use of permanent marker to deposit a protection layer against FIB damage in TEM specimen preparation

Permanent marker deposition (PMD), which creates permanent writing on an object with a permanent marker, was investigated as a method to deposit a protection layer against focused ion beam damage. PMD is a simple, fast and cheap process. Further, PMD is excellent in filling in narrow and deep trenches, enabling damage‐free observation of high aspect ratio structures with atomic resolution in transmission electron microscopy (TEM). The microstructure, composition, gap filling ability and planarization of the PMD layer were studied using dual beam focused ion beam, transmission electron microscopy, energy dispersive X‐ray spectroscopy and electron energy loss spectroscopy. It was found that a PMD layer is basically an amorphous carbon structure, and that such a layer should be at least 65 nm thick to protect a surface against 30 keV focused ion beam damage. We suggest that such a PMD layer can be an excellent protection layer to maintain a pristine sample structure against focused ion beam damage during transmission electron microscopy specimen preparation.     

FIB-induced damage in silicon

The damage created in silicon transmission electron microscope specimens prepared using a focused ion beam miller is assessed using cross‐sections of trenches milled under different beam conditions. Side‐wall damage consists of an amorphous layer formed by direct interaction with the energetic gallium ion beam; a small amount of implanted gallium is also detected. By contrast, bottom‐wall damage layers are more complex and contain both amorphous films and crystalline regions that are richer in implanted gallium. More complex milling sequences show that redeposition of milled material, enriched in gallium, can occur depending on the geometry of the mill employed. The thickness of the damage layers depends strongly on beam energy, but is independent of beam current. Monte Carlo modelling of the damage formed indicates that recoil silicon atoms contribute significantly to the damaged formed in the specimen.     

A technique for improved focused ion beam milling of cryo-prepared life science specimens

The combination of focused ion beam and scanning electron microscopy with a cryo‐preparation/transfer system allows specimens to be milled at low temperatures. However, for biological specimens in particular, the quality of results is strongly dependent on correct preparation of the specimen surface. We demonstrate a method for deposition of a protective, planarizing surface layer onto a cryo‐sample, enabling high‐quality cross‐sectioning using the ion beam and investigation of structures at the nanoscale.     

一种滚动导轨磨削加工尺寸在线测量仪

针对滚动导轨磨削加工尺寸的在线测量问题,介绍了一种导轨磨削加工尺寸在线测量仪,该仪器以半导体激光器配合光电二极管作工件位置检测器、整体切割的平行弹簧,配合音圈电机作测头驱动器和双电感位移传感器作偏差尺寸检测器,采用相对测量法,配合数据采集卡、工控机和上层控制软件,能对导轨磨削尺寸进行在线检测。     

Novel techniques of preparing TEM samples for characterization of irradiation damage

Focus ion beam preparation of transmission electron microscopy (TEM) samples has become increasingly popular due to the relative ease of extraction of TEM foils from specific locations within a larger sample. However the sputtering damage induced by Ga ion bombardment in focus ion beam means that traditional electropolishing may be a preferable method. First, we describe a special electropolishing method for the preparation of irregular TEM samples from ex‐service nuclear reactor components, spring‐shaped spacers. This method has also been used to prepare samples from a nonirradiated component for a TEM in situ heavy ion irradiation study. Because the specimen size is small (0.7 × 0.7 × 3 mm), a sandwich installation is adopted to obtain high quality polishing. Second, we describe some modifications to a conventional TEM cross‐section sample preparation method that employs Ni electroplating. There are limitations to this method when preparing cross‐section samples from either (1) metals which are difficult to activate for electroplating, or (2) a heavy ion irradiated foil with a very shallow damage layer close to the surface, which may be affected by the electroplating process. As a consequence, a novel technique for preparing cross‐section samples was developed and is described.     

Application of the focused ion beam technique in aerosol science: detailed investigation of selected, airborne particles

The focused ion beam technique was used to fabricate transmission electron microscope lamellas of selected, micrometre‐sized airborne particles. Particles were sampled from ambient air on Nuclepore polycarbonate filters and analysed with an environmental scanning electron microscope. A large number of particles between 0.6 and 10 µm in diameter (projected optical equivalent diameter) were detected and analysed using computer‐controlled scanning electron microscopy. From the resulting dataset, where the chemistry, morphology and position of each individual particle are stored, two particles were selected for a more detailed investigation. For that purpose, the particle‐loaded filter was transferred from the environmental scanning electron microscope to the focused ion beam, where lamellas of the selected particles were fabricated. The definition of a custom coordinate system enabled the relocation of the particles after the transfer. The lamellas were finally analysed with an analytical transmission electron microscope. Internal structure and elemental distribution maps of the interior of the particles provided additional information about the particles, which helped to assign the particles to their sources. The combination of computer‐controlled scanning electron microscopy, focused ion beam and transmission electron microscopy offers new possibilities for characterizing airborne particles in great detail, eventually enabling a detailed source apportionment of specific particles. The particle of interest can be selected from a large dataset (e.g. based on chemistry and/or morphology) and then investigated in more detail in the transmission electron microscope.     

Effect of gallium focused ion beam milling on preparation of aluminium thin foils

Focussed ion beam milling has greatly extended the utility of the atom probe and transmission electron microscope because it enables sample preparation with a level of dimensional control never before possible. Using focussed ion beam it is possible to extract the samples from desired and very specific locations. The artefacts associated with this sample preparation method must also be fully understood. In this work, issues specifically relevant to the focussed ion beam milling of aluminium alloys are presented. After using the focussed ion beam as a sample preparation technique it is evident that gallium will concentrate in three areas of the sample: on the surface, on grain boundaries and at interphase boundaries. This work also shows that low-energy Ar ion nanomilling is potentially quite effective for removing gallium implantation layers and gallium from the internal surfaces of aluminium thin foils.     

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.     

Simple technique for high‐throughput marking of distinguishable micro‐areas for microscopy

Today's (nano)‐functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 μm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin‐film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron‐beam evaporation which ensures good line‐of‐sight deposition. This inexpensive high‐throughput method has several advantageous over other marking techniques such as focused ion‐beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion‐beam sectioning to obtain a thin lamellar of a particular pre‐selected area.     

Quantitative comparison of segmentation algorithms for FIB‐SEM images of porous media

Focused ion beam tomography has proven to be capable of imaging porous structures on a nano‐scale. However, due to shine‐through artefacts, common segmentation algorithms often lead to severe dislocation of individual structures in z‐direction. Recently, a number of approaches have been developed, which take into account the specific nature of focused ion beam‐scanning electron microscope images for porous media. In the present study, we analyse three of these approaches by comparing their performance based on simulated focused ion beam‐scanning electron microscope images. Performance is measured by determining the amount of misclassified voxels as well as the fidelity of structural characteristics. Based on this analysis we conclude that each algorithm has certain strengths and weaknesses and we determine the scenarios for which each approach might be the best choice     

Towards the imaging of Weibel–Palade body biogenesis by serial block face‐scanning electron microscopy

Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face‐scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three‐dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre‐scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three‐dimensional analysis. Here, we explore focused ion beam facilitated serial block face‐scanning electron microscopy to study the endothelial cell–specific storage organelles, the Weibel–Palade bodies, during their biogenesis at the Golgi apparatus. Weibel–Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face‐scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel–Palade bodies and Golgi stacks which became conceivable in large‐scale three‐dimensional data. We demonstrate that serial block face‐scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.