Electron microscopy of nanoemulsions: an essential tool for characterisation and stability assessment

The characterisation of pharmaceutical formulations by microscopic techniques is essential to obtain reliable data about the actual morphology of the system. Since the size range of colloidal drug delivery systems has long ago reached the lower end of the nanometer scale, classical light microscopy has been replaced by electron microscopy techniques which provide sufficient resolution for the visualisation of nano-sized structures. Indeed, the superior resolution and methodological versatility of electron microscopy has rendered this technique an indispensable tool for the analysis of nanoemulsions. Microscopic analysis of these lipid-based drug delivery systems with particle sizes in the lower submicron range provides critical information about the size, shape and internal structure of the emulsion droplets. Moreover, surfactant aggregates such as liposomes or multilamellar structures which remain unnoticed during particle size measurements can be detected in this fashion. This review provides a brief overview about both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques which have been employed to characterise nanoemulsions. Of special interest are sophisticated cryo techniques of sample preparation for both TEM and SEM which deliver high-quality images of nanoemulsions in their natural state. An overview about the instrumentation and sample preparation for all presented methods is given. Important practical aspects, sources of error and common artefacts as well as recent methodological advances are discussed. Selected examples of electron microscopic studies of nanoemulsions are presented to illustrate the potential of this technique to reveal detailed and specific information.     

Low voltage high-resolution SEM (LVHRSEM) for biological structural and molecular analysis

High-resolution scanning electron microscopy (HRSEM) is being used increasingly to gain new insights into three-dimensional organization of biological structure, macromolecular complexes and interactions of cellular components as well as isolated cell organelles. Modern scanning electron microscopes (SEMs) combined with adequate sample preparation can now provide resolution comparable with that achieved using transmission electron microscopes (TEMs) down to 2-5 nm for biological material. The versatility of the instrument and new sample preparation techniques have allowed detailed analysis of chromosomes, cytoskeletal components, virus and other biological material that has not been possible with TEM. The present review addresses resolution and specific specimen preparations for HRSEM, and highlights the importance of specimen preparation and choice of methods to achieve optimal results for proteins, macromolecular complexes and subcellular structures using low voltage HRSEM (LVHRSEM).     

Exploring the third dimension: Volume electron microscopy comes of age

Groundbreaking advances in volume electron microscopy and specimen preparation are enabling the 3-dimensional visualisation of specimens with unprecedented detail, and driving a gratifying resurgence of interest in the ultrastructural examination of cellular systems. Serial section techniques, previously the domain of specialists, are becoming increasingly automated with the development of systems such as the automatic tape-collecting ultramicrotome, and serial blockface and focused ion beam scanning electron microscopes. These changes are rapidly broadening the scope of biomedical studies to which volume electron microscopy techniques can be applied beyond the brain. Further innovations in microscope design are also in the pipeline, which have the potential to enhance the speed and quality of data collection. The recent introduction of integrated light and electron microscopy systems will revolutionise correlative light and volume electron microscopy studies, by enabling the sequential collection of data from light and electron imaging modalities without intermediate specimen manipulation. In doing so, the acquisition of comprehensive functional information and direct correlation with ultrastructural details within a 3-dimensional reference space will become routine. The prospects for volume electron microscopy are therefore bright, and the stage is set for a challenging and exciting future.     

The application of graphene as a sample support in transmission electron microscopy

Transmission electron microscopy has witnessed rampant development and surging point resolution over the past few years. The improved imaging performance of modern electron microscopes shifts the bottleneck for image contrast and resolution to sample preparation. Hence, it is increasingly being realized that the full potential of electron microscopy will only be realized with the optimization of current sample preparation techniques. Perhaps the most recognized issues are background signal and noise contributed by sample supports, sample charging and instability. Graphene provides supports of single atom thickness, extreme physical stability, periodic structure, and ballistic electrical conductivity. As an increasing number of applications adapting graphene to their benefit emerge, we discuss the unique capabilities afforded by the use of graphene as a sample support for electron microscopy.     

Strain mapping of semiconductor specimens with nm-scale resolution in a transmission electron microscope

The last few years have seen a great deal of progress in the development of transmission electron microscopy based techniques for strain mapping. New techniques have appeared such as dark field electron holography and nanobeam diffraction and better known ones such as geometrical phase analysis have been improved by using aberration corrected ultra-stable modern electron microscopes. In this paper we apply dark field electron holography, the geometrical phase analysis of high angle annular dark field scanning transmission electron microscopy images, nanobeam diffraction and precession diffraction, all performed at the state-of-the-art to five different types of semiconductor samples. These include a simple calibration structure comprising 10-nm-thick SiGe layers to benchmark the techniques. A SiGe recessed source and drain device has been examined in order to test their capabilities on 2D structures. Devices that have been strained using a nitride stressor have been examined to test the sensitivity of the different techniques when applied to systems containing low values of deformation. To test the techniques on modern semiconductors, an electrically tested device grown on a SOI wafer has been examined. Finally a GaN/AlN superlattice was tested in order to assess the different methods of measuring deformation on specimens that do not have a perfect crystalline structure. The different deformation mapping techniques have been compared to one another and the strengths and weaknesses of each are discussed.     

A High Resolution,Hard X-ray Bio-imaging Facility at SSRL

The old saying that seeing is believing has particular resonance for studying biological cells and tissues. Since 1677, when Anton van Leeuwenhoek used a simple light microscope to discover single cell organisms, scientists have relied on structural information obtained from microscopes with improving capabilities to advance the understanding of how biological systems work. Optical and electron microscopes are essential for many of these important discoveries and have been widely employed in biomedical research laboratories. However, various limitations exist in these microscopy techniques. We describe below how the new X-ray imaging facility at the Stanford Synchrotron Radiation Laboratory (SSRL), based on an Xradia nano-XCT full-field transmission X-ray microscope (TXM), can provide complementary and unique capabilities to the current microscopy methods for studying complex biological systems.     

Structure analysis of soluble proteins using electron crystallography

Electron crystallography as a structural determination technique has grown dramatically in use over recent years. Improvements in microscopes, equipment, practical techniques, computation facilities and image processing methods are reflected in the increasing number of near-atomic resolution structures that have been published.In this review we shall summarize the techniques involved in structure determination of soluble proteins using electron crystallography. Many soluble protein structures have been investigated in this manner over the past two decades. Here we present several examples where a variety of approaches have been used to gradually increase the information obtained.     

Quantitative electron diffraction: From atoms to bonds

The development of new transmission electron microscopes with energy-filtering capability together with the increase in computer power over the past few years has enabled electron diffraction from inorganic crystals to become a quantitative and accurate science. With the introduction of new techniques and data analysis and with the advantage of a nanometre-sized probe, electron diffraction now rivals X-ray and neutron methods in many aspects of crystallography and solid-state physics. In this article, we have discussed two new developments which highlight the progress made in this area. First, a new method for ab-initio structure determination is explained and an example given to show its success. Second, energy-filtered diffraction patterns are used to refine the scattering potential of a crystal so that the bonding charge density can be reconstructed.     

Probing bacterial interactions: integrated approaches combining atomic force microscopy, electron microscopy and biophysical techniques

Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.     

Case studies on the application of high-resolution electron channelling contrast imaging – investigation of defects and defect arrangements in metallic materials

In 1967, Coates discovered the electron channelling contrast of backscattered electrons (BSEs) in scanning electron microscopy, and by this the possibility to investigate arrangements of lattice defects in deformed microstructures of materials. Since that time, a straightforward development of the scanning electron microscopes as well as of the electron channelling contrast technique took place. Nowadays, the performance of scanning electron microscopes is high enough that the resolution of electron channelling contrast imaging (ECCI) micrographs is comparable with conventional bright field transmission electron microscopy (TEM) micrographs. In the first part of the present paper, a historical review on the development of the ECCI technique starting from its discovery more than 45 years ago up to the combination with other advanced methods of scanning electron microscopy like electron backscatter diffraction or high-resolution selected area channelling patterning in the last few years is given. Major important investigations using this technique for the visualization of individual lattice defects like stacking faults (SFs) and dislocations or dislocation arrangements are chronologically summarized. The second part demonstrates that nowadays, ECCI micrographs taken in high-resolution scanning electron microscopes can be called high-resolution ECCI (HR-ECCI). It is shown that the resolution of individual SFs and dislocations in the HR-ECCI micrographs is comparable to that of conventional TEM (about 15 nm defect image width). Furthermore, the paper is demonstrating that HR-ECCI micrographs can be obtained for various types of materials after different mechanical loadings and different grain sizes ranging from large grain size of 500 μm (cast steel) down to less than 2 μm (γ-TiAl).     

Two-dimensional crystallogenesis of transmembrane proteins

Two-dimensional crystallogenesis is a crucial step in the long road that leads to the determination of macromolecules structure via electron crystallography. The necessity of having large and highly ordered samples can hold back the resolution of structural works for a long time, and this, despite improvements made in electron microscopes or image processing. Today, finding good conditions for growing two-dimensional crystals still rely on either "biocrystallo-cooks" or on lucky ones. The present review presents the field by first describing the different crystals that one can encounter and the different crystallisation methods used. Then, the effects of different components (such as protein, lipids, detergent, buffer, and temperature) and the different methods (dialysis, hydrophobic adsorption) are discussed. This discussion is punctuated by correspondences made to the world of three-dimensional crystallogenesis. Finally, a guide for setting up 2D crystallogenesis experiments, built on the discussion mentioned before, is proposed to the reader. More than giving recipes, this review is meant to open up the discussions in this field.     

Characterisation of colloidal drug delivery systems from the naked eye to Cryo-FESEM

Poly(ethylcyanoacrylate) nanoparticles prepared by interfacial polymerisation on the basis of microemulsions were prepared in this study and both colloidal systems, nanoparticles and microemulsions, were analysed by visual observation and several microscopic techniques. Phase boundaries for the microemulsions of the two pseudoternary systems ethyloleate, polyoxyethylene 20 sorbitan mono-oleate/sorbitan monolaurate and water with and without butanol as a cosurfactant were determined by visual observation of the samples. Microemulsions containing liquid crystals were determined by polarisation light microscopy. Using freeze-fracture transmission electron microscopy and Cryo-field emission scanning electron microscopy the type of microemulsion (w/o droplet, bicontinuous, solution) was characterised. Nanoparticles prepared from the different types of microemulsion were additionally observed by conventional scanning electron microscopy. The size of the nanoparticles obtained from electron microscopy was in good agreement with particle sizing techniques (photon correlation spectroscopy) from earlier studies and no morphological differences could be observed in particles prepared from the different types of microemulsions. Cryo-field emission scanning electron microscopy proved to be a most valuable technique in the visualisation of the colloidal systems as samples could be observed close to their natural state.     

Test object for emission electron microscope

The resolution of emission electron microscopes approaches some nanometers, which leads to the need for new test objects. Microfields, which are almost always present at the sample surface, deform the trajectories of electrons forming the image. This leads to a distortion of the emission electron microscopy image and a decrease of lateral resolution. We propose a test object, where the influence of microfields conditioned by contact potential differences is compensated by a specially shaped relief of the sample surface.     

Laser cleaning of steel for paint removal

Paint removal is an important part of steel processing for marine and offshore engineering. For centuries, a blasting techniques have been widely used for this surface preparation purpose. But conventional blasting always has intrinsic problems, such as noise, explosion risk, contaminant particles, vibration, and dust. In addition, processing wastes often cause environmental problems. In recent years, laser cleaning has attracted much research effort for its significant advantages, such as precise treatment, and high selectivity and flexibility in comparison with conventional cleaning techniques. In the present study, we use this environmentally friendly technique to overcome the problems of conventional blasting. Processed samples are examined with optical microscopes and other surface characterization tools. Experimental results show that laser cleaning can be a good alternative candidate to conventional blasting.     

The high-voltage electron microscope

The advantages of using very high voltages for electron microscopy are reviewed, in respect of greater specimen thickness and reduced chromatic aberration. For polymers and possibly with living material, the lower radiation damage in a given thickness is also valuable. The special problems in the design and construction of microscopes operating up to 1 Mv are discussed. We are still in a stage of rapid development, and some possibilities for the future are outlined. The main areas of application of high voltage microscopes are beginning to be defined; some examples are given of what has been done with the Toulouse and Cambridge microscopes to date.     

Visualisation of results from LES of combustion

We examine the role of visualisation in the context of LES simulations of premixed turbulent combustion. The physical processes involved in premixed turbulent combustion are extremely complex, and the modelling of both the turbulence (via LES) and the combustion (via flame-wrinkling models) is difficult. Appropriate visualisation is required to understand the behaviour of the models, and ultimately to understand better the flow processes which are important in many industrial applications. We examine visualisations of two specific cases; simple flame kernel growth in a box of turbulence, and combustion behind a backward-facing step. A number of visualisation techniques are used to produce results that are similar to experimentally determined Schlieren and Mie photography for the flame kernel. In addition, isosurfaces of the reaction regress variable coloured by the laminar flame speed and sub-grid wrinkling are also plotted in an attempt to gain deeper insight into the physics of turbulent combustion in the context of these particular cases. Finally we discuss the role of the WWW in the continuing development of scientific visualisation techniques.     

Superior tribological properties of an amorphous carbon film with a graphite-like structure

Amorphous carbon films with high sp² concentrations are deposited by unbalanced magnetron sputtering with a narrow range of substrate bias voltage. Field emission scanning electron microscopes (FESEMs), high resolution transmission electron microscopes (HRTEMs), atomic force microscopes (AFMs), the Raman spectrometers, nano-indentation, and tribometers are subsequently used to characterize the microstructures and the properties of the resulting films. It is found that the present films are dominated by the sp² sites. However, the films demonstrate a moderate hardness together with a low internal stress. The high hardness of the deposited film originates from the crosslinking of the sp² clusters by the sp³ sites. The presence of the graphite-like clusters in the film structure may be responsible for the low internal stress. What is more important is that the resulting films show excellent tribological properties with high load capacity and excellent wear resistance in humid atmospheres. The relationship between the microstructure determined by the deposition condition and the film characteristic is discussed in detail.     

Correlative microscopy: providing new understanding in the biomedical and plant sciences

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological, structural and chemical information that exceeds what is possible with any single technique. As a variety of complementary microscopy approaches rather than a specific type of instrument, correlative microscopy has blossomed in recent years as researchers have recognised that it is particularly suited to address the intricate questions of the modern biological sciences. Specialised technical developments in sample preparation, imaging methods, visualisation and data analysis have also accelerated the uptake of correlative approaches. In light of these advances, this critical review takes the reader on a journey through recent developments in, and applications of, correlative microscopy, examining its impact in biomedical research and in the field of plant science. This twin emphasis gives a unique perspective into use of correlative microscopy in fields that often advance independently, and highlights the lessons that can be learned from both fields for the future of this important area of research.     

A new ultra‐high‐vacuum variable temperature and high‐magnetic‐field X‐ray magnetic circular dichroism facility at LNLS

X‐ray magnetic circular dichroism (XMCD) is one of the most powerful tools for investigating the magnetic properties of different types of materials that display ferromagnetic behavior. Compared with other magnetic‐sensitive techniques, XMCD has the advantage of being element specific and is capable of separating the spin and magnetic moment contributions associated with each element in the sample. In samples involving, for example, buried atoms, clusters on surfaces or at interfaces, ultrathin films, nanoparticles and nanostructures, three experimental conditions must be present to perform state‐of‐the‐art XMCD measurements: high magnetic fields, low temperatures and an ultra‐high‐vacuum environment. This paper describes a new apparatus that can be easily installed at different X‐ray and UV beamlines at the Brazilian Synchrotron Light Laboratory (LNLS). The apparatus combines the three characteristics described above and different methods to measure the absorption signal. It also permits in situ sample preparation and transfer to another chamber for measurement by conventional surface science techniques such as low‐energy electron diffraction (LEED), reflection high‐energy electron diffraction (RHEED), X‐ray photoelectron spectroscopy (XPS) and X‐ray photoelectron diffraction (XPD). Examples are given of XMCD measurements performed with this set‐up on different materials.     

Aberration correction and its automatic control in scanning electron microscopes

An automatic aberration correction method has been implemented in scanning electron microscopes (SEM). Necessity of the automatic aberration correction is discussed. The procedure of the automatic aberration correction is explained in detail, where deconvolution techniques are used in order to extract probe information from SEM images. Due to the precise digitization and the usage of proper combinations of correction fields, linearity has been found between the amplitude of each aberration and the corresponding field strength. Experimental results are shown which demonstrate that the aberrations are corrected automatically by a linear feedback control method. After the automatic aberration correction, the image quality has been improved drastically.