Revealing local variations in nanoparticle size distributions in supported catalysts: a generic TEM specimen preparation method

The specimen preparation method is crucial for how much information can be gained from transmission electron microscopy (TEM) studies of supported nanoparticle catalysts. The aim of this work is to develop a method that allows for observation of size and location of nanoparticles deposited on a porous oxide support material. A bimetallic Pt‐Pd/Al₂O₃ catalyst in powder form was embedded in acrylic resin and lift‐out specimens were extracted using combined focused ion beam/scanning electron microscopy (FIB/SEM). These specimens allow for a cross‐section view across individual oxide support particles, including the unaltered near surface region of these particles. A site‐dependent size distribution of Pt‐Pd nanoparticles was revealed along the radial direction of the support particles by scanning transmission electron microscopy (STEM) imaging. The developed specimen preparation method enables obtaining information about the spatial distribution of nanoparticles in complex support structures which commonly is a challenge in heterogeneous catalysis.     

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.     

Characterization of the μ and P phase precipitates in the CMSX‐4 single crystal superalloy

A combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning‐transmission electron microscopy (STEM) using high‐angle annular‐dark‐field (HAADF) imaging, focussed ion beam‐ scanning electron microscopy (FIB‐SEM) tomography, selected area electron diffraction with beam precession (PED), as well as spatially resolved energy‐dispersive X‐ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS), was used to investigate topologically close‐packed (TCP) phases, occurring in the CMSX‐4 superalloy subjected to high temperature annealing and creep deformation. Structural and chemical analyses were performed to identify the TCP phases and provide information concerning the compositional partitioning of elements between them. The results of SEM and FIB‐SEM tomography revealed the presence of merged TCP particles, which were identified by TEM and PED analysis as coprecipitates of the μ and P phases. Inside the TCP particles that were several micrometres in size, platelets of alternating μ and P phases of nanometric width were found. The combination of STEM‐HAADF imaging with spatially resolved EDS and EELS microanalysis allowed determination of the significant partitioning of the constituent elements between the μ and P phases.     

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.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Effects of the Ar ions pre‐amorphization of Si substrateon interface mixing of Fe/Si bilayers

Ion beam mixing of Fe/Si bilayers, induced by 100 keV ⁴⁰Arions at room temperature was investigated. Rutherford backscattering spectroscopy (RBS), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied for structural characterization. The main focus of this study was on the influence of the substrate structure on interface mixing. The influence of the substrate structure is due to the two classes of irradiated bilayers, Fe thin films deposited on crystalline or pre‐amorphized Si substrates. An about 76% higher efficiency of atomic transport across the pre‐amorphized Fe/a‐Si interface as compared to that of Fe/c‐Si bilayers was observed.     

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.     

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.     

Ultrastructure of submandibular salivary glands of mouse: TEM and HRSEM observations

The fine structure of submandibular glands of mouse were analyzed using light microscopy (LM), high resolution scanning electron microscopy (HRSEM), and transmission electron microscopy (TEM) methods. For LM, the specimens were embedded in Spurr resin, stained by toluidin blue solutions. For TEM, the tissues of submandibular salivary glands were fixed with modified Karnovsky solution and postfixed with osmium tetroxide. For HRSEM, the tissues were fixed with 2% osmium tetroxide solution in 1/15M sodium phosphate buffer (pH 7.4). The samples were immersed successively in dymethylsulphoxide and freeze cracked. The maceration was made in diluted osmium tetroxide for 24-48 h. The samples were examined by high resolution scanning electron microscopy. The intracellular components of acinar and ductal cells revealed clearly the Golgi apparatus, rough endoplasmic reticulum, secretory granules, and mitochondria. The end bulbs of Golgi lamellae and flattened cisterns of rough endoplasmic reticulum showed the luminal surface. A few mitochondria were identified intermingling between the rough endoplasmic reticulum and the mitochondriales cristae in three-dimensional HRSEM images. Secretory granules were numerous and presented different sizes. Small granules of ribosomes were attached on cistern surface, measuring 20-25 nm in diameter. Numerous arranged microvilli were found on the luminal surface of secretory canaliculus. The contact surfaces of acinar cells revealed complicated interdigitations by cytoplasmic processes. The mitochondria of duct cells were disposed vertically and surrounded by basal infoldings of plasma membranes. Basement membrane showed a spongy-like structure having an irregular surface with various strands and meshes of fine collagen fibrils.     

Analytical transmission electron microscopy and electron diffraction for characterization of multiphase Ni-P-Ti surface layer on Ti-6Al-4V alloy

The microstructure, chemical and phase composition of the hard Ni‐P‐Ti layer formed on the Ti‐6Al‐4V alloy after duplex surface treatment were investigated by light microscopy, X‐ray diffraction, scanning electron microscopy and analytical/high‐resolution transmission electron microscopy. These investigations showed that the improved mechanical and tribological properties of the surface‐treated alloy were related to the presence of a multilayered microstructure containing several phases from the Ni‐Ti‐P‐Al system.     

Anisotropic Friction of the Ventral Scales in the Snake Lampropeltis getula californiae

Since the ventral body side of snakes is in almost continuous contact with the substrate during undulating locomotion, their skin is presumably adapted to generate high friction for propulsion and low friction to slide along the substrate. In this study, the microstructure of ventral scales was analyzed using scanning electron microscopy, atomic force microscope and confocal laser scanning microscopy. Dynamic friction was investigated by a microtribometer. The ventral scales demonstrated anisotropic frictional properties. To analyze the role of the stiffness of underlying layers on the frictional anisotropy, two different types of scale cushioning (hard and soft) were tested. To estimate frictional forces of the skin surface on rough substrates, additional measurements with a rough surface were performed. Frictional anisotropy for both types of scale cushioning and rough surfaces was revealed. However, for both types of surface roughness, the anisotropy was stronger expressed in the soft-cushioned sample. This effect could be caused by (1) the stronger interaction of the microstructure with the substrate in soft-cushioned samples due to larger real contact area with the substrate and (2) the composite character of the skin of this snake species with embedded, highly ordered fiber-like structures, which may cause anisotropy in material properties.     

Spin‐coated epoxy resin embedding technique enables facile SEM/FIB thickness determination of porous metal oxide ultra‐thin films

A facile nonsubjective method was designed to measure porous nonconductive iron oxide film thickness using a combination of a focused ion beam (FIB) and scanning electron microscopy. Iron oxide films are inherently nonconductive and porous, therefore the objective of this investigation was to optimize a methodology that would increase the conductivity of the film to facilitate high resolution imaging with a scanning electron microscopy and to preserve the porous nature of the film that could potentially be damaged by the energy of the FIB. Sputter coating the sample with a thin layer of iridium before creating the cross section with the FIB decreased sample charging and drifting, but differentiating the iron layer from the iridium coating with backscattered electron imaging was not definitive, making accurate assumptions of the delineation between the two metals difficult. Moreover, the porous nature of the film was lost due to beam damage following the FIB process. A thin layer plastication technique was therefore used to embed the porous film in epoxy resin that would provide support for the film during the FIB process. However, the thickness of the resin created using conventional thin layer plastication processing varied across the sample, making the measuring process only possible in areas where the resin layer was at its thinnest. Such variation required navigating the area for ideal milling areas, which increased the subjectivity of the process. We present a method to create uniform thin resin layers, of controlled thickness, that are ideal for quantifying the thickness of porous nonconductive films with FIB/scanning electron microscopy.     

Towards correlative imaging of plant cortical microtubule arrays: combining ultrastructure with real-time microtubule dynamics

There are a variety of microscope technologies available to image plant cortical microtubule arrays. These can be applied specifically to investigate direct questions relating to array function, ultrastructure or dynamics. Immunocytochemistry combined with confocal laser scanning microscopy provides low resolution "snapshots" of cortical microtubule arrays at the time of fixation whereas live cell imaging of fluorescent fusion proteins highlights the dynamic characteristics of the arrays. High-resolution scanning electron microscopy provides surface detail about the individual microtubules that form cortical microtubule arrays and can also resolve cellulose microfibrils that form the innermost layer of the cell wall. Transmission electron microscopy of the arrays in cross section can be used to examine links between microtubules and the plasma membrane and, combined with electron tomography, has the potential to provide a complete picture of how individual microtubules are spatially organized within the cortical cytoplasm. Combining these high-resolution imaging techniques with the expression of fluorescent cytoskeletal fusion proteins in live cells using correlative microscopy procedures will usher in an radical change in our understanding of the molecular dynamics that underpin the organization and function of the cytoskeleton.     

Transmission electron microscopy with a liquid flow cell

The imaging of microscopic structures at nanometre-scale spatial resolution in a liquid environment is of interest for a wide range of studies. Recently, a liquid flow transmission electron microscopy (TEM) holder equipped with a microfluidic cell has been developed and shown to exhibit flow of nanoparticles through an electron transparent viewing window. Here we demonstrate the application of the flow cell system for both scanning and conventional transmission electron microscopy imaging of immobilized nanoparticles with a resolution of a few nanometres in liquid water of micrometre thickness. The spatial resolution of conventional TEM bright field imaging is shown to be limited by chromatic aberration due to multiple inelastic scattering in the water, and we demonstrate that the liquid in the cell can be displaced by a gas phase that forms under intense electron irradiation. Our data suggest that under appropriate conditions, TEM imaging with a liquid flow cell is a promising method for understanding the in situ behaviour of nanoscale structures in a prescribed and dynamically changing chemical environment.     

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM – SEM)

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.     

An appraisal of ultramicrotomy, FIBSEM and cryogenic FIBSEM techniques for the sectioning of biological cells on titanium substrates for TEM investigation

Ultramicrotomy, focused ion beam scanning electron microscopy (FIBSEM) and cryogenic FIBSEM (cryo-FIBSEM) techniques, as developed for the controlled cross-sectioning of mesenchymal stem cells (MSCs) and human osteoblasts (HObs) on titanium (Ti) substrates for transmission electron microscopy (TEM) investigation, are compared. Conventional ultramicrotomy has been used to section cells on Ti-foil substrates embedded in resin, but significant problems with cell detachment using this technique restricted its general applicability. Conventional FIBSEM 'lift-out' procedures were found to be effective for the preparation of uniform sections of fixed and dehydrated cell/Ti specimens, but the control of cell staining remains an issue. Cryo-FIBSEM procedures used with an 'H-bar' sample geometry enabled the sectioning of fixed and hydrated cell/Ti specimens, but issues remain over ion beam-induced artefacts and control of frost on the sample foils.     

Microstructural analysis of a FeAl/quasicrystal-based composite prepared using a focused ion beam miller

A composite consisting of a brittle multiphase matrix containing both an Al-based quasicrystalline phase (ψ) and an ordered body centred cubic phase (β) and a relatively ductile ordered body centred cubic intermetallic FeAl phase has been developed as an abrasive wear-resistant coating material. It is applied as a 500 μm thick layer onto stainless steel substrates through plasma spray processing. The microstructure of such materials can be readily examined by optical and scanning electron microscopy, but the inherent difficulty of preparing transmission electron microscope (TEM) samples has inhibited higher resolution studies. However, the relatively recent development of the focused ion beam (FIB) miller as a tool in materials science provides a method ideal for the preparation of TEM specimens of these materials. In this study a coating consisting of a mixture of an Al–Cu–Fe based quasicrystal and FeAl+Cr was deposited on to a 304 stainless steel substrate. TEM specimens were prepared using a FIB and subjected to detailed microstructural characterization. The structure consisted of elongated bands of a FeAl phase about 100 nm in width and several micrometres in length, which enclosed more equiaxed regions about 1 μm in diameter that consisted of fine mixtures of quasicrystal and two Al-Fe-Cu phases isostructurally related to FeAl.     

Transmission Electron Microscopy Analysis of Mo–W–S–Se Film Sliding Contact Obtained by Using Focused Ion Beam Microscope and In Situ Microtribometer

To better understand the fundamentals of solid lubrication, microstructural analyses on the wear scar surface and contact interface of Mo–W–S–Se composite films produced by pulsed laser deposition were completed. Focused ion beam (FIB), transmission electron microscopy (TEM), and X-ray energy dispersive spectroscopy were employed to study the cross-sectional microstructure and chemistry of wear scars. In particular, a novel microtribometer was built for in situ tribological measurements within a FIB microscope. The sliding tip was welded in contact to the wear scar surface on the film under load by re-deposition of sputtering materials from the FIB cut of the tip. Using this technique, cross-sectional TEM specimens were prepared precisely at the contact point without tip/film separation. Here, the in situ FIB microtribometer is critically important for retaining the microstructure of lubricant films as formed at the sliding contact interface between the tip and film without separation. It provides the unique ability to stop sliding, section the contact, and reveal microstructural changes to that contact without disrupting the sliding interface. The cross-sectional TEM measurements were performed on the sliding contact interface for both the regions in contact and just past contact, and both the reorientation and recrystallization of lubricant films were revealed.     

Correlation of two‐photon in vivo imaging and FIB/SEM microscopy

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two‐photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long‐term two‐photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three‐dimensional high‐resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.     

Multiple imaging techniques demonstrate the manipulation of surfaces to reduce bacterial contamination and corrosion

Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. The complementarity of the microscopy methods, scanning electron microscopy, electron probe microanalysis and atomic force microscopy assessed and correlated form and function of the surface modifications. Stainless steel disks (1 cm in diameter) were laser‐cut from the same sheets of stainless steel and treated by electropolishing or left untreated for controls. Each treatment was analysed separately using each technique. First, the disks were examined by visual inspection and electron probe microanalysis for surface characteristics and elemental composition, respectively. Aliquots of bacterial suspensions (saline rinses of poultry carcasses from a commercial broiler processing plant) were then diluted in broth and monitored for growth by spectrophotometry. Stainless steel disks (1 cm in diameter) were added and the cultures were grown to sufficient density to allow attachment of bacterial cells to test surfaces. Relative differences in the surface morphology shown by atomic force microscopy, including Z ranges, roughness and other measurements, corresponded by treatment with the differences in reduction of bacterial counts shown by scanning electron microscopy. A model of wet‐processing conditions tested the effects of corrosive treatment of surfaces. Less bacterial attachment occurred after corrosive treatment on controls and electropolished samples. Electropolishing significantly reduced bacterial numbers and the effects of corrosive action compared to the controls. Thus, the multiple imaging techniques showed that engineered changes on stainless steel surfaces improved the resistance of the surface finish to bacterial attachment, biofilm formation, and corrosive action.