An in-lens Schottky field emission scanning electron microscope (SEM) combined with a transmission electron microscope (TEM)-type cold-stage and a chromium (Cr) sputter-coating system was developed to rapidly prepare and cryo-image biological specimens to attain accurate nanometer-level structural information. High-resolution topographic images at high primary magnification ([eg ]200,000 times) were digitally recorded with very short dwell times and without beam damage. Plunge freezing in ethane, followed by fracturing, Cr coating, and in-lens cryo-high-resolution scanning electron microscope (HRSEM) imaging directly revealed macromolecular features of yeast cells, platelets, and cell-free elastin analogues. The 'vitreous' nature of bulk water in its solid state appeared featureless in cryo-HRSEM images, suggesting that if ice crystals were present they would be [el ]2–3 nm (the approximate instrument resolution on cryo-specimens). Compared to technically difficult and indirect freeze-fracture TEM replicas, cryo-HRSEM samples are fully hydrated, unfixed, noncryoprotected specimens immersed in featureless ice. The time necessary to cryo-immobilize the specimen and record the image is <3 hr. The hexagonal arrays of intramembrane particles on the protoplasmic face of yeast cells and differences in surface morphology between thrombin-stimulated and quiescent platelets were assessed. A clear interface line between collapsed elastin fibril lacework and vitreous lakes was commonly observed. These experiments demonstrate the feasibility of this technique to rapidly evaluate macromolecular features in cryofixed cells and cell-free systems.

A new closed cell is presented for in situ X-ray ptychography which allows studies under gas flow and at elevated temperature. In order to gain complementary information by transmission and scanning electron microscopy, the cell makes use of a Protochips E-chipTM which contains a small, thin electron transparent window and allows heating. Two gold-based systems, 50 nm gold particles and nanoporous gold as a relevant catalyst sample, were used for studying the feasibility of the cell. Measurements showing a resolution around 40 nm have been achieved under a flow of synthetic air and during heating up to temperatures of 933 K. An elevated temperature exhibited little influence on image quality and resolution. With this study, the potential of in situ hard X-ray ptychography for investigating annealing processes of real catalyst samples is demonstrated. Furthermore, the possibility to use the same sample holder for ex situ electron microscopy before and after the in situ study underlines the unique possibilities available with this combination of electron microscopy and X-ray microscopy on the same sample.

Interfaces play critical roles in materials and are usually both structurally and compositionally complex microstructural features. The precise characterization of their nature in three-dimensions at the atomic scale is one of the grand challenges for microscopy and microanalysis, as this information is crucial to establish structure–property relationships. Atom probe tomography is well suited to analyzing the chemistry of interfaces at the nanoscale. However, optimizing such microanalysis of interfaces requires great care in the implementation across all aspects of the technique from specimen preparation to data analysis and ultimately the interpretation of this information. This article provides critical perspectives on key aspects pertaining to spatial resolution limits and the issues with the compositional analysis that can limit the quantification of interface measurements. Here, we use the example of grain boundaries in steels; however, the results are applicable for the characterization of grain boundaries and transformation interfaces in a very wide range of industrially relevant engineering materials.

This work describes the application and usefulness of the focused ion beam (FIB) technique for the preparation of transmission electron microscopy (TEM) samples from metal matrix composite materials. Results on an Al/diamond composite, manufactured by the squeeze casting infiltration process, were chosen for demonstration. It is almost impossible to prepare TEM specimens of this material by any other conventional method owing to the presence of highly inhomogeneous phases and reinforcement diamond particles. The present article gives a detailed account of the salient features of the FIB technique and its operation. One of the big advantages is the possibility to prepare site-specific TEM specimens with high spatial resolution. The artifacts occurring during the specimen preparation, for example, Ga-ion implantation, curtain effects, amorphous layers, bending of the lamella, or different milling behaviors of the materials have been discussed. Furthermore, TEM examination of the specimens prepared revealed an ultrafine amorphous layer of graphite formed at the interface between the Al and diamond particles that may affect the interfacial properties of the composite materials. This may not have been feasible without the successful application of the FIB technique for production of good quality site-specific TEM specimens.

This article focuses on the development of a transparent and uniform understanding of possibilities for three-dimensional (3D) imaging in scanning transmission and confocal electron microscopes (STEMs and SCEMs), with an emphasis on the annular dark-field STEM (ADF-STEM), bright-field SCEM (BF-SCEM), and ADF-SCEM configurations. The incoherent imaging approximation and a 3D linear imaging model for ADF-STEM are reviewed. A 3D phase contrast model for coherent-SCEM as well as a pictorial way to find boundaries of information transfer in reciprocal space are reviewed and applied to both BF- and ADF-SCEM to study their 3D point spread functions and contrast transfer functions (CTFs). ADF-STEM is capable of detecting the depths of dopant atoms in amorphous materials but can fail for crystalline materials when channeling substantially modifies the electron propagation. For the imaging of extended (i.e., nonpointlike) features, ADF-STEM and BF-SCEM exhibit strong elongation artifacts due to the missing cone of information. ADF-SCEM shows an improvement over ADF-STEM/BF-SCEM due to its differential phase contrast eliminating slowly varying backgrounds, an effect that partially suppresses the elongation artifacts. However, the 3D CTF still has a cone of missing information that will result in some residual feature elongation as has been observed in A. Hashimoto et al., J Appl Phys160(8), 086101 (2009).

The capability to perform liquid in situ transmission electron microscopy (TEM) experiments provides an unprecedented opportunity to examine the real-time processes of physical and chemical/electrochemical reactions during the interaction between metal surfaces and liquid environments. This work describes the requisite steps to make the technique fully analytical, from sample preparation, through modifications of the electrodes, characterization of electrolytes, and finally to electrochemical corrosion experiments comparing in situ TEM to conventional bulk cell and microcell configurations.

Atom probe is a powerful technique for studying the composition of nano-precipitates, but their morphology within the reconstructed data is distorted due to the so-called local magnification effect. A new technique has been developed to mitigate this limitation by characterizing the distribution of the surrounding matrix atoms, rather than those contained within the nano-precipitates themselves. A comprehensive chemical analysis enables further information on size and chemistry to be obtained. The method enables new insight into the morphology and chemistry of niobium carbonitride nano-precipitates within ferrite for a series of Nb-microalloyed ultra-thin cast strip steels. The results are supported by complementary high-resolution transmission electron microscopy.

Cs correctors have revolutionized transmission electron microscopy (TEM) in that they substantially improve point resolution and information limit. The object information is found sharply localized within 0.1 nm, and the intensity image can therefore be interpreted reliably on an atomic scale. However, for a conventional intensity image, the object exit wave can still not be detected completely in that the phase, and hence indispensable object information is missing. Therefore, for example, atomic electric-field distributions or magnetic domain structures cannot be accessed. Off-axis electron holography offers unique possibilities to recover completely the aberration-corrected object wave with uncorrected microscopes and hence we would not need a Cs-corrected microscope for improved lateral resolution. However, the performance of holography is affected by aberrations of the recording TEM in that the signal/noise properties (“phase detection limit”) of the reconstructed wave are degraded. Therefore, we have realized off-axis electron holography with a Cs-corrected TEM. The phase detection limit improves by a factor of four. A further advantage is the possibility of fine-tuning the residual aberrations by a posteriori correction. Therefore, a combination of both methods, that is, Cs correction and off-axis electron holography, opens new perspectives for complete TEM analysis on an atomic scale.

Scanning transmission electron microscope (STEM) images of three-dimensional (3D) samples were simulated. The samples consisted of a micrometer(s)-thick substrate and gold nanoparticles at various vertical positions. The atomic number (Z) contrast as obtained via the annular dark-field detector was generated. The simulations were carried out using the Monte Carlo method in the CASINO software (freeware). The software was adapted to include the STEM imaging modality, including the noise characteristics of the electron source, the conical shape of the beam, and 3D scanning. Simulated STEM images of nanoparticles on a carbon substrate revealed the influence of the electron dose on the visibility of the nanoparticles. The 3D datasets obtained by simulating focal series showed the effect of beam broadening on the spatial resolution and on the signal-to-noise ratio. Monte Carlo simulations of STEM imaging of nanoparticles on a thick water layer were compared with experimental data by programming the exact sample geometry. The simulated image corresponded to the experimental image, and the signal-to-noise levels were similar. The Monte Carlo simulation strategy described here can be used to calculate STEM images of objects of an arbitrary geometry and amorphous sample composition. This information can then be used, for example, to optimize the microscope settings for imaging sessions where a low electron dose is crucial for the design of equipment, or for the analysis of the composition of a certain specimen.

A culture's confluency is a fundamental measure in the field of biology, and routine quantification of confluence in cell culture protocols, biological assays and tissue engineering work is important. However, current techniques for obtaining confluency are either subjective, destructive, not simple enough, or time-consuming. We developed an image processing method for automated confluency measurement from a single microscope image without any chemical staining. To demonstrate utility we monitored the confluency of three cell types: NIH3T3 fibroblasts, C2C12 myoblasts, and 3T3L1 pre-adipocytes for 5 days, twice a day. The captured micrographs had different and uneven illumination, the cell types varied in cell-to-background contrast, and the confluency ranged between 10% and 100%. Despite these variable conditions, our method was shown to be practical, economic, and easy to implement, providing quantitative confluency measurements over time in each culture case. The method is hence suitable for routine automatic determination of confluency to standardize handling of cells, achieve reproducibility across trials, and improve accuracy in experimental outcome measures.

Automated inclusion microanalysis in steel samples by computer-based scanning electron microscopy provides rapid quantitative information on micro-inclusion distribution, composition, size distribution, morphology, and concentration. Performing the analysis at a lower accelerating voltage (10 kV), rather than the generally used 20 kV, improves analysis accuracy and may improve spatial resolution, but at the cost of a smaller backscattered electron signal and potentially smaller rate of generation of characteristic X-rays. These effects were quantified by simulation and practical measurements.

Electron back scatter diffraction (EBSD) mapping and indexing has rapidly come into widespread use. However, inadequate attention has been paid to the details of the method. Many of the algorithms in current use were chosen because they were the first ones that were found to work, rather than because they were optimum. Results of systematic study are presented. It is shown that more than one method can successfully correct a sampling artifact, that there is an optimum binning ratio, that Gaussian filtering provides an alternative to “butterfly convolution,” that better alternatives for mapping image quality than those in current use are available, and that saving all the original patterns is practical and advantageous.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Focused ion beam (FIB) induced damage in nanocrystalline Al thin films has been characterized using advanced transmission electron microscopy techniques. Electron tomography was used to analyze the three-dimensional distribution of point defect clusters induced by FIB milling, as well as their interaction with preexisting dislocations generated by internal stresses in the Al films. The atomic structure of interstitial Frank loops induced by irradiation, as well as the core structure of Frank dislocations, has been resolved with aberration-corrected high-resolution annular dark-field scanning TEM. The combination of both techniques constitutes a powerful tool for the study of the intrinsic structural properties of point defect clusters as well as the interaction of these defects with preexisting or deformation dislocations in irradiated bulk or nanostructured materials.

Ion channeling contrast (iCC) and electron channeling contrast (eCC) are caused by variation in signal resulting from changes in the angle of the incident beam and the crystal lattice with respect to the target. iCC is directly influenced by the incident ion range in crystalline materials. The ion range is larger for low-index crystal orientated grains, resulting in the emission of fewer secondary electrons at the surface yielding a lower signal. Ions are stopped closer to the surface for off-axis grains, resulting in the emission of many secondary electrons yielding a higher signal. Conversely, backscattered electrons (BSEs) are the primary contribution to eCC. BSEs are diffracted or channeled to form an electron channeling pattern (ECP). The BSE emission of the ECP peaks when the electron beam is normal to the surface of an on-axis grain, and therefore a bright signal is observed. Thus, iCC and eCC images yield inverse contrast behavior for on-axis oriented grains. Since there is a critical angle associated with particle channeling, accurately determining grain boundary locations require the acquisition of multiple images obtained at different tilt conditions.

Ocular surface diseases (OSDs) represent a widely investigated field of research given their growing incidence and the negative impact on quality of life. During OSDs, cytokines generated by damaged epithelia trigger and deregulate the lymphoid cells composing the eye-associated lymphoid tissues, inducing an immune-mediated chronic inflammation that amplifies and propagates the disease during time. The conjunctiva-associated lymphoid tissue (CALT), given its particular position that permits immune cells covering the cornea, might play a crucial role in the development of OSDs. Despite the recognized inflammatory role of mucosa-associated lymphoid tissues in other stations taking contact with the external environment (gut or bronchus), CALT did not gain the deserved consideration. In the last years, the diffusion of the in vivo confocal microscopy (IVCM) stimulated the interest to CALT, especially in dry eye, ocular allergy, and glaucoma. Though the initial stimuli were different, IVCM documented similar changes, represented by increased lymphoid cells within the diffuse layer, follicles and interfollicular spaces. These findings, which need to be validated by immunohistology, support the CALT stimulation during OSDs. However, while an involvement of the CALT in OSDs is hypothesizable, the exact role of this structure in their pathogenesis remains unclear and warrants further investigations.

The dictionary-based approach to the indexing of diffraction patterns is applied to electron channeling patterns (ECPs). The main ingredients of the dictionary method are introduced, including the generalized forward projector (GFP), the relevant detector model, and a scheme to uniformly sample orientation space using the “cubochoric” representation. The GFP is used to compute an ECP “master” pattern. Derivative free optimization algorithms, including the Nelder–Mead simplex and the bound optimization by quadratic approximation are used to determine the correct detector parameters and to refine the orientation obtained from the dictionary approach. The indexing method is applied to poly-silicon and shows excellent agreement with the calibrated values. Finally, it is shown that the method results in a mean disorientation error of 1.0° with 0.5° SD for a range of detector parameters.

Off-axis electron holography is used to measure electrostatic potential profiles across a silicon p-n junction, which has been prepared for examination in the transmission electron microscope (TEM) in two different specimen geometries using focused ion beam (FIB) milling. Results are obtained both from a conventional unbiased FIB-milled sample and using a novel sample geometry that allows a reverse bias to be applied to an FIB-milled sample in situ in the TEM. Computer simulations are fitted to the results to assess the effect of TEM specimen preparation on the charge density and the electrostatic potential in the thin sample.