Electron microscopy of pharmaceutical systems

During the last decades, the focus of research in pharmaceutical technology has steadily shifted towards the development and optimisation of nano-scale drug delivery systems. As a result, electron microscopic methods are increasingly employed for the characterisation of pharmaceutical systems such as nanoparticles and microparticles, nanoemulsions, microemulsions, solid lipid nanoparticles, different types of vesicles, nanofibres and many more. Knowledge of the basic properties of these systems is essential for an adequate microscopic analysis. Classical transmission and scanning electron microscopic techniques frequently have to be adapted for an accurate analysis of formulation morphology, especially in case of hydrated colloidal systems. Specific techniques such as environmental scanning microscopy or cryo preparation are required for their investigation. Analytical electron microscopic techniques such as electron energy-loss spectroscopy or energy-dispersive X-ray spectroscopy are additional assets to determine the elemental composition of the systems, but are not yet standard tools in pharmaceutical research. This review provides an overview of pharmaceutical systems of interest in current research and strategies for their successful electron microscopic analysis. Advantages and limitations of the different methodological approaches are discussed and recent findings of interest are presented.     

Atomic force microscopy and transmission electron microscopy of biocompatible magnetic fluids: A comparative analysis

The present study provides a comparative analysis of the size dispersity of magnetic nanoparticles (MNPs) within magnetic fluids as obtained from atomic force microscopy (AFM) and transmission electron microscopy (TEM). Whereas the mean particle diameter obtained from the AFM data presented a reduction of about 34% as compared to the value obtained from the TEM data, the standard deviation obtained from the AFM data is twice the value found from the TEM data. Similarities and differences in the size dispersity parameters are discussed in terms of sample preparation and tip characteristics. A two-dimensional mode for the deposition of the MNPs on top of the mica substrate is discussed as well.     

Determination of particle size in multiphase blends by different methods

Different methods for characterizing the morphology of multiphase blends were applied to a blend of thermoplastic polyurethane with 20 wt% polypropylene as the dispersed phase. Optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and light scattering were compared. The microscopy methods were evaluated with respect to their suitability for quantitative image analysis for determination of the particle size distribution. Comparison of the particle size distributions revealed that the dependence of the measured particle size on the method of preparation and technique was not very pronounced. The main difference resulted from cutting the particles outside their maximum diameter. The measured particle sizes determined with methods that analyze the whole particles, such as SEM on separated particles and laser light scattering, are larger than those measured on cut specimens. The factor 4/π valid in monodisperse systems for the ratio between the real particle size and that measured on sections was found also to be applicable to this polydisperse blend system. Although light micros-copy requires the least preparation efforts, it is a reliable method for this blend system.     

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).     

Recent Developments of Crystallographic Analysis Methods in the Scanning Electron Microscope for Applications in Metallurgy

The field of metallurgy has greatly benefited from the development of electron microscopy over the last two decades. Scanning electron microscopy (SEM) has become a powerful tool for the investigation of nano- and microstructures. This article reviews the complete set of tools for crystallographic analysis in the SEM, i.e., electron backscatter diffraction (EBSD), transmission Kikuchi diffraction (TKD), and electron channeling contrast imaging (ECCI). We describe recent relevant developments in electron microscopy, and discuss the state-of-the-art of the techniques and their use for analyses in metallurgy. EBSD orientation measurements provide better angular resolution than spot diffraction in TEM but slightly lower than Kikuchi diffraction in TEM, however, its statistical significance is superior to TEM techniques. Although spatial resolution is slightly lower than in TEM/STEM techniques, EBSD is often a preferred tool for quantitative phase characterization in bulk metals. Moreover, EBSD enables the measurement of lattice strain/rotation at the sub-micron scale, and dislocation density. TKD enables the transmitted electron diffraction analysis of thin-foil specimens. The small interaction volume between the sample and the electron beam enhances considerably the spatial resolution as compared to EBSD, allowing the characterization of ultra-fine-grained metals in the SEM. ECCI is a useful technique to image near-surface lattice defects without the necessity to expose two free surfaces as in TEM. Its relevant contributions to metallography include deformation characterization of metals, including defect visualization, and dislocation density measurements. EBSD and ECCI are mature techniques, still undergoing a continuous expansion in research and industry. Upcoming technical developments in electron sources and optics, as well as detector instrumentation and software, will likely push the border of performance in terms of spatial resolution and acquisition speed. The potential of TKD, combined with EDS, to provide crystallographic, chemical, and morphologic characterizations of nano-structured metals will surely be a valuable asset in metallurgy.     

Synthesis by sol–gel process,structural and optical properties of nanoparticles of zinc oxide doped vanadium

We report the elaboration of vanadium-doped ZnO nanoparticles prepared by a sol–gel processing technique. In our approach, the water for hydrolysis was slowly released by esterification reaction followed by a supercritical drying in ethyl alcohol. Vanadium doping concentration of 10 at.% has been investigated. After treatment in air at different temperatures, the obtained nanopowder was characterised by various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and photoluminescence (PL). Analysis by scanning electron microscopy at high resolution shows that the grain size increases with increasing temperature. Thus, in the case of thermal treatment at 500 °C in air, the powder with an average particle size of 25 nm shows a strong luminescence band in the visible range. The intensity and energy position of the obtained PL band depends on the temperature measurement increase. The mechanism of this emission band is discussed.     

Turbiscan多重光散射法研究纳米银溶胶的分散稳定性

以硝酸银为银源、间苯二酚为还原剂、聚乙烯吡咯烷酮为保护剂通过化学还原法制得纳米银颗粒,通过离心洗涤等操作洗掉多余的反应物,将其分别超声分散于水、无水乙醇和乙二醇中获得0.2 Wt%不同体系的纳米银溶胶。利用激光粒度分析仪、透射电子显微镜、扫描电子显微镜和同步热分析仪对纳米银颗粒进行表征并测定纳米银溶胶中的银含量。激光粒度分析仪的粒度分析结果表明,实验制得的纳米银颗粒的粒径在100 nm左右,且粒径分布均一。透射电子显微镜和扫描电子显微镜的结果进一步证实纳米银颗粒的粒径在纳米尺度范围且粒径均匀。采用Turbiscan多重光散射法研究了不同体系纳米银溶胶的分散稳定性,分析了导致纳米银溶胶不稳定的主要因素。研究发现：影响纳米银溶胶分散稳定性的主要因素包括颗粒粒径的变化和颗粒的迁移。对于水相体系,样品池中间部分背散射光强度随时间变化不大,样品池底部和顶部背散射光强度有较大变化,说明其稳定性的主要影响因素是纳米银颗粒的迁移,颗粒粒径变化影响不大;对于乙醇和乙二醇体系,样品池中间部分、底部和顶部的背散射光强度均有明显变化,说明颗粒粒径的变化和颗粒的迁移对体系稳定性均有一定程度的影响;最后通过比较三个体系的稳定性动力学指数,得到体系稳定性由高到低依次为乙二醇、水和乙醇。     

Structural features of laser ablation particulate from Si target, as revealed by focused ion beam technology

Heterogeneity in laser-induced particle structures was investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of individual particles micro-machined using focused ion beam (FIB). The primary particle size distribution spanned over three orders of magnitude, i.e., in the range 10 nm–10 m with few larger secondary objects. The particulate larger than 0.5 m often resulted from particle–particle aggregation, mostly upon a spherical core, seldom in chain-like structures. The core of these fractal aggregates was found to be polycrystalline. The heterogeneity of the particles with respect to structure and chemistry is surely of importance for elemental analysis using laser ablation as sample introduction technique. PACS 39.30.+w; 61.46.+w; 81.20.Rg     

Integration of correlative Raman microscopy in a dualbeam FIB SEM

We present an integrated confocal Raman microscope in a focused ion beam scanning electron microscope (FIB SEM). The integrated system enables correlative Raman and electron microscopic analysis combined with focused ion beam sample modification on the same sample location. This provides new opportunities, for example the combination of nanometer resolution with Raman advances the analysis of sub‐diffraction‐sized particles. Further direct Raman analysis of FIB engineered samples enables in situ investigation of sample changes. The Raman microscope is an add‐on module to the electron microscope. The optical objective is brought into the sample chamber, and the laser source, and spectrometer are placed in a module attached onto and outside the chamber. We demonstrate the integrated Raman FIB SEM function with several experiments. First, correlative Raman and electron microscopy is used for the investigation of (sub‐)micrometer‐sized crystals. Different crystals are identified with Raman, and in combination with SEM the spectral information is combined with structurally visible polymorphs and particle sizes. Analysis of sample changes made with the ion beam is performed on (1) structures milled in a silicon substrate and (2) after milling with the FIB on an organic polymer. Experiments demonstrate the new capabilities of an integrated correlative Raman–FIB–SEM. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Challenges and strategies of surface modification of electrogalvanized coatings for electron microscopy analysis

Despite wide usage of electrogalvanized coatings in various applications, characterization studies on their micro/crystal structure, and the understanding of how they correspondingly affect the properties, such as corrosion, are rather limited. This is mainly attributed to some difficulties in preparing and examining the zinc coating layers, owing to their intrinsically low corrosion resistance and refined nano-scaled crystallite size. This study aims to examine such challenges systematically and propose some mitigation strategies. Particularly, sample preparation processes, including surface finishing for metallography and sample thinning processes are explored. Furthermore, a range of electron microscopy techniques, including scanning electron microscopy (SEM), electron back scattered diffractometry (EBSD), and transmission electron microscopy (TEM) are investigated in relation to the achievable clarity of microstructural details of electrogalvanized coatings.     

强流脉冲电子束诱发纯镍表层纳米结构的形成机制

利用强流脉冲电子束（HCPEB）技术对多晶纯镍进行了表面处理,并采用扫描电镜和透射电镜对强流脉冲电子束诱发的表面及亚表面的微观组织结构进行了分析.实验结果表明,HCPEB辐照后表面熔化,形成了深度约为2 μm的重熔层,快速的凝固使重熔层中形成晶粒尺寸约为80 nm的纳米结构.位于轰击表面下方5—15 μm深度范围内强烈塑性变形引起的位错墙和其内部的亚位错墙结构是该区域的主要结构特征.这些缺陷结构通过互相交割细化晶粒,最终导致尺寸约为10 nm的纳米晶粒的形成. 关键词： 强流脉冲电子束 纳米结构 多晶纯镍 位错墙     

Quantitative atomic-scale analysis of interface structures: transmission electron microscopy and local density functional theory

Transmission electron microscopy (TEM) and local density functional theory (LDFT) are combined to analyze the microscopic structure of the rhombohedral twin interface in alpha-Al2O3. LDFT provides interfacial energetics and atomic and electronic structures for three competing models. With high-resolution TEM the atomic structure at the interface is imaged quantitatively along two orthogonal zone axes. Electron energy loss spectroscopy in TEM with nanoscale spatial resolution yields the interfacial electronic structure. Both experiments confirm the theoretically preferred model quantitatively.     

The morphology of submicronsized core-shell latex particles: an electron microscopy study

The core-shell structure of a range of acrylic-acrylic latexes has been investigated by combining different specimen preparation methods with transmission electron microscopy (TEM), dark-field scanning transmission electron microscopy (DSTEM) and low-voltage scanning electron microscopy (LV-SEM), including the first reported use of LV-SEM to observe composite latex particles at ambient and subambient temperatures. Spin-coating of liquid latex dispersions directly onto TEM grids or SEM stubs is shown to be a relatively straightforward mean of avoiding film formation during specimen preparation. In conjunction with double staining techniques, it has been found to be particularly convenient for characterizing the fine structure of particles with diameters down to below 100 nm.     

Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of ∼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.     

Scanning electron microscopy study on nanoemulsions and solid lipid nanoparticles containing high amounts of ceramides

Ceramides are the most important intercellular lipids of the stratum corneum, regulating the barrier function of the skin and participating as second signal messenger in stress-induced apoptosis. The high lipophilicity of ceramides presents a pharmacological problem. In order to overcome this problem two lipophilic delivery systems were used for the incorporation of the ceramides: (1) nanoemulsions (NE) and (2) solid lipid nanoparticles (SLN). The influence of the incorporation of ceramides on the particle shape, size and Polydispersity Index was investigated by photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). The results showed that NE can incorporate larger amounts of ceramides than SLN (up to 23.2% and 5% of lipid matrix, respectively) without any significant alteration on the morphology of the dispersed particles. The incorporation of higher amounts of ceramides into SLN, leads to anisometric platelet-like formations that are known to be caused by the transition of triglycerides from - to β-mesomorph. The results of this study can be useful for the design of appropriate delivery systems and for further pharmacological evaluations.     

空心Fe_3O_4纳米微球的制备及超顺磁性

采用水热控制合成法,以六水三氯化铁、柠檬酸三钠和尿素为原料,聚丙烯酰胺为稳定剂, 200?C下反应12 h制备得到了超顺磁性空心Fe_3O_4纳米微球.通过X射线衍射仪、扫描电子显微镜、透射电子显微镜对样品的结构和形貌进行表征,并采用振动样品磁强计测试了样品的磁性能.结果表明:所得样品为具有尖晶石结构的Fe_3O_4纳米微球,尺寸为160 nm左右,呈分等级结构,即整个微球由粒径约18 nm的初级晶粒自组装堆叠而成;室温下表现为典型的超顺磁性,且饱和磁化强度为73.3 emu/g (1 emu/g=1 A·m~2/kg),这种高饱和磁化强度可以由其初级晶粒晶化程度高且粒径较大以及这种特殊的二次自组装结构进行解释.这种Fe_3O_4纳米微球为疏松多孔的空心球状结构,具有粒径分布均匀、分散性良好和超顺磁性的特点,在药物靶向输运和肿瘤热疗中有潜在的应用.     

原子尺度材料三维结构、磁性及动态演变的透射电子显微学表征

原子表征与操控是实现原子制造必须突破的物理瓶颈之一.像差校正电子显微学方法因其优异的空间分辨率,为实现原子精细制造提供了有力的表征手段.因此,利用电子显微学手段,在原子尺度对原子制造的材料及器件进行三维结构和性能的协同表征,对于深入理解原子水平材料操控的物理机理具有非常重要的意义.纳米团簇及纳米颗粒是原子制造材料与器件研究的主要对象之一,具有丰富的物理化学性质和较高的可操纵性.本文探讨纳米团簇/颗粒结构三维定量表征、使役条件下纳米团簇/颗粒结构演变定量表征、纳米颗粒/晶粒结构-成分-磁性协同定量表征等诸多方法与实例,阐明了电子显微学表征手段的突破和发展为实现精细控制的原子制造材料提供了坚实基础.     

Direct Synthesis of ZIF-8 on Transmission Electron Microscopy Grids Allows Structure Analysis and 3D Reconstruction

The first example of layer-by-layer growth of a metal–organic framework (MOF) directly on transmission electron microscopy (TEM) grids is described. ZIF-8 is deposited on thin amorphous carbon films and subjected to a structure analysis by (scanning) TEM ((S)TEM). This method serves as a two-in-one synthesis and TEM sample-preparation technique and allows straightforward analysis of ZIF-8 crystallites. Artifacts resulting from sample preparation are completely avoided by this approach. The morphological properties, crystal structure, and the chemical composition of the material are investigated with high spatial resolution by a variety of methods of (analytical) electron microscopy. Furthermore, the incorporation of metallic nanoparticles in ZIF-8 by integrating a corresponding step into the layer-by-layer deposition process is examined. The formation of ZIF-8 crystals on the film proceeds as under the absence of nanoparticle-forming synthesis steps. However, the nanoparticles rather cover the supporting amorphous carbon film than being incorporated in the ZIF-8 material. This information cannot be obtained from standard characterization techniques but requires the application of analytical (S)TEM techniques.     

Focused ion beam preparation of samples for X‐ray nanotomography

The preparation of hard material samples with the necessary size and shape is critical to successful material analysis. X‐ray nanotomography requires that samples are sufficiently thin for X‐rays to pass through the sample during rotation for tomography. One method for producing samples that fit the criteria for X‐ray nanotomography is focused ion beam/scanning electron microscopy (FIB/SEM) which uses a focused beam of ions to selectively mill around a region of interest and then utilizes a micromanipulator to remove the milled‐out sample from the bulk material and mount it on a sample holder. In this article the process for preparing X‐ray nanotomography samples in multiple shapes and sizes is discussed. Additionally, solid‐oxide fuel cell anode samples prepared through the FIB/SEM technique underwent volume‐independence studies for multiple properties such as volume fraction, average particle size, tortuosity and contiguity to observe the characteristics of FIB/SEM samples in X‐ray nanotomography.