Imaging Si nanoparticles embedded in SiO(2) layers by (S)TEM-EELS

Fabrication of systems in which Si nanoparticles are embedded in a thin silica layer is today mature for non-volatile memory and opto-electronics applications. The control of the different parameters (position, size and density) of the nanoparticles population is a key point to optimize the properties of such systems. A review of dedicated transmission electron microscopy (TEM) methods, which can be used to measure these parameters, is presented with an emphasis on those relying on electron energy-loss spectroscopy (EELS). Defocused bright-field imaging can be used in order to determine topographic information of a whole assembly of nanoparticles, but it is not efficient for looking at individual nanoparticles. High-resolution electron imaging or dark-field imaging can be of help in the case of crystalline particles but they always provide underestimated values of the nanocrystals population. EELS imaging in the low-energy-loss domain around the Si plasmon peak, which gives rise to strong signals, is the only way to visualize all Si nanoparticles within a silica film and to perform reliable size and density measurements. Two complementary types of experiments are investigated and discussed more extensively: direct imaging with a transmission electron microscope equipped with an imaging filter (EFTEM) and indirect imaging from spectrum-imaging data acquired with a scanning transmission electron microscope equipped with a spectrometer (STEM-PEELS). The direct image (EFTEM) and indirect set of spectra (STEM-PEELS) are processed in order to deliver images where the contribution of the silica matrix is minimized. The contrast of the resulting images can be enhanced with adapted numerical filters for further morphometric analysis. The two methods give equivalent results, with an easier access for EFTEM and the possibility of a more detailed study of the EELS signatures in the case of STEM-PEELS. Irradiation damage in such systems is also discussed.     

What atomic resolution annular dark field imaging can tell us about gold nanoparticles on (1 1 0)

Annular dark field scanning transmission electron microscopy imaging was recently applied to a catalyst consisting of gold nanoparticles on TiO₂ (1 1 0), showing directly that the gold atoms in small nanoparticles preferentially attach to specific sites on the TiO₂ (1 1 0) surface. Here, through simulation, a parameter exploration of the imaging conditions which maximise the visibility of such nanoparticles is presented. Aberration correction, finite source size and profile imaging are all considered while trying to extracting the maximum amount of information from a given sample. Comment is made on the role of the thermal vibration of the atoms in the nanoparticle, the magnitude of which is generally not known a priori but which affects the visibility of the nanoparticles in this imaging mode.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample preparation protocol was developed to enable imaging of cells and gold nanoparticles with a conventional below lens scanning electron microscopes. The negative influence of 'charging' on the quality of scanning electron microscopes' images could be limited by deposition of biological cells on a conductive (gold) surface. The novel protocol enabled high-resolution scanning electron microscopes' imaging of small clusters and individual gold nanoparticles on uncoated cell surfaces. Gold nanoparticles could be counted on cancer cells with automated routines.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

基于迭代补偿的纳米粒子磁化信号检测方法研究

磁粒子成像是一种无创成像技术,通过检测磁粒子示踪剂磁化信号,表征其浓度分布图像。在实际检测中,检测线圈的感应信号包含激励磁场信号与磁性纳米粒子磁化信号。将激励信号从感应电压中去除,获取粒子信号是磁性粒子成像信号检测需要解决的关键问题。针对磁性纳米粒子成像信号检测中激励磁场耦合消除方法进行研究,设计平面梯度检测线圈,并提出迭代补偿控制方法,消除激励磁场耦合,实现磁性纳米粒子磁化信号检测。仿真计算与实验测量的结果表明,对于不同检测模型,所提出的检测方法均可以完成粒子信号检测。该方法获得的粒子信号的信噪比是原有信号消去检测方法的2.2倍,与滤波方法相比信噪比提高到1.3倍,激励磁场耦合衰减可达到34 dB。     

Nanoparticle embedded chitosan film for agglomeration free TEM images

Transmission electron microscopy (TEM) is a very useful and commonly used microscopy technique, used especially for the characterization of nanoparticles. However, the identification of the magnetic nanoparticle could be thought problematic in TEM analysis, due to the fact that the magnetic nanoparticles are usually form aggregates on the TEM grid to form bigger particles generating higher stability. This prevents to see exact shape and size of each nanoparticle. In order to overcome this problem, a simple process for the formation of well‐dispersed nanoparticles was conducted, by covering chitosan film on the unmodified copper grid, it was said to result in aggregation‐free TEM images. It is also important to fix the magnetic nanoparticles on the TEM grids, due to possible contamination of TEM filament which is operated under high vacuum conditions. The chitosan film matrix also helps to protect the TEM filament from contact with magnetic nanoparticles during the imaging process. The proposed procedure offers a quick method to fix the nanoparticles in a conventional copper TEM grid and chitosan matrix prevents agglomeration of nanoparticles, and thus getting TEM images showing well‐dispersed individual nanoparticles.     

有序金纳米棒阵列的超分辨成像

显微成像技术受限于光学成像系统的衍射极限，无法分辨亚波长尺度的结构。通过饱和散射抑制成像技术已经实现了单个纳米颗粒的超分辨成像，但是涉及到纳米颗粒集合，需要考虑纳米颗粒间的耦合作用。利用超越衍射极限的双光束方法，可以在有序金纳米棒阵列上实现远场超分辨光学成像。本文设计了纳米棒长径比为2的5×5金纳米棒阵列，通过矢量光场理论和热扩散理论计算了金纳米棒阵列在连续波激光下的热分布，并模拟了双光束激光即脉冲激发光和连续波抑制光下的散射成像。仿真结果显示，连续波激光能够有效抑制金纳米棒阵列对脉冲激光的散射，双光束方法实现了80 nm横向特征尺寸的超分辨成像。     

Nanometer-resolution electron microscopy through micrometers-thick water layers

Scanning transmission electron microscopy (STEM) was used to image gold nanoparticles on top of and below saline water layers of several micrometers thickness. The smallest gold nanoparticles studied had diameters of 1.4 nm and were visible for a liquid thickness of up to 3.3 μm. The imaging of gold nanoparticles below several micrometers of liquid was limited by broadening of the electron probe caused by scattering of the electron beam in the liquid. The experimental data corresponded to analytical models of the resolution and of the electron probe broadening as function of the liquid thickness. The results were also compared with Monte Carlo simulations of the STEM imaging on modeled specimens of similar geometry and composition as used for the experiments. Applications of STEM imaging in liquid can be found in cell biology, e.g., to study tagged proteins in whole eukaryotic cells in liquid and in materials science to study the interaction of solid:liquid interfaces at the nanoscale.     

Imaging cobalt nanoparticles by amplitude modulation atomic force microscopy: comparison between low and high amplitude solutions

In many situations of interest amplitude modulation AFM is characterized by the coexistence of two solutions with different physical properties. Here, we compare the performance of those solutions in the imaging of cobalt nanoparticles. We show that imaging with the high amplitude solution implies an irreversible deformation of the nanoparticles while repeated imaging with the low solution does not produce noticeable changes in the nanoparticles. Theoretical simulations show that the maximum tip-surface force in the high amplitude solution is about 14nN while in the low amplitude solution is about -4nN. We attribute the differences in the high and low amplitude images to the differences in the exerted forces on the sample.     

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.     

Mesoporous iron oxide nanoparticles prepared by polyacrylic acid etching and their application in gene delivery to mesenchymal stem cells

Novel monodisperse mesoporous iron oxide nanoparticles (m‐IONPs) were synthesized by a postsynthesis etching approach and characterized by electron microscopy. In this approach, solid iron oxide nanoparticles (s‐IONPs) were first prepared following a solvothermal method, and then etched anisotropically by polyacrylic acid to form the mesoporous nanostructures. MTT cytotoxicity assay demonstrated that the m‐IONPs have good biocompatibility with mesenchymal stem cells (MSCs). Owing to their mesoporous structure and good biocompatibility, these monodisperse m‐IONPs were used as a nonviral vector for the delivery of a gene of vascular endothelial growth factor (VEGF) tagged with a green fluorescence protein (GFP) into the hard‐to‐transfect stem cells. Successful gene delivery and transfection were verified by detecting the GFP fluorescence from MSCs using fluorescence microscopy. Our results illustrated that the m‐IONPs synthesized in this work can serve as a potential nonviral carrier in gene therapy where stem cells should be first transfected and then implanted into disease sites for disease treatment. Microsc. Res. Tech. 76:936–941, 2013. © 2013 Wiley Periodicals, Inc.     

Nanoscale characterization of gold nanoparticles created by in situ reduction at a polymeric surface

Transmission Electron Microscopy is used as a quantitative method to measure the shapes, sizes and volumes of gold nanoparticles created at a polymeric surface by three different in situ synthesis methods. The atomic number contrast (Z‐contrast) imaging technique reveals nanoparticles which are formed on the surface of the polymer. However, with certain reducing agents, the gold nanoparticles are additionally found up to 20 nm below the polymer surface. In addition, plan‐view high‐angle annular dark‐field scanning transmission electron microscopy images were statistically analyzed on one sample to measure the volume, height and effective diameter of the gold nanoparticles and their size distributions. Depth analysis from high‐angle annular dark‐field scanning transmission electron microscopy micrographs also gives information on the dominant shape of the nanoparticles.     

分子影像技术活体追踪移植干细胞的研究进展

干细胞具有分化、再生能力,通过体外扩增和体内移植,可以治疗各种组织坏损和退化性疾病(如心脑血管疾病、脑脊髓外伤和糖尿病等),具有极大的应用前景,是目前国际、国内的研究热点。利用核素显像、磁共振成像和光成像等分子影像技术,通过体外直接标记、报告基因或功能显示等追踪策略,可以显示干细胞在活体内的分布和变化,明确其最终归宿和产生的功能。合理选择这些分子影像技术和追踪策略,或通过互补结合,将有助于阐明干细胞在活体内的作用机制和相关的影响因素,指导临床干细胞治疗抉择和疗效评估。     

Imaging nanoparticle‐algae interactions in three dimensions using Cytoviva microscopy

Plasmonic resonances of metal‐based nanoparticles are increasingly used for ultrasensitive imaging assays. In this context, the Cytoviva^TM microscopy platform has greatly gained in popularity. In essence, Cytoviva is an optimized dark field microscope that permits detection of particles down to a few nanometers in size. A significant limitation of Cytoviva up to now has been that it only provided for single plane imaging. The datasets produced by this technique therefore only show a partial view of the sample – not ideally suited to analysis. Here we explain how to overcome this limitation by mounting the Cytoviva condenser on an automated microscope with Z‐scanning capability. Our method allows three‐dimensional mapping of nanoparticles in their full three‐dimensional cellular context. We apply this technique to study the interaction of silver and cerium dioxide nanoparticles with cells of the green alga, Pseudokirchneriella subcapitata, a system of significant environmental relevance because algae underlie much of the aquatic food chain. Our objective was to develop a technique to visualize in detail the interaction of nanoparticles with cells in three dimensions, such that one may, for example, determine whether a particular nanoparticle is inside a cell, at its very surface, or at a distance from it.     

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.     

A method for correcting the effect of specimen drift on coherent diffractive imaging

Coherent diffractive imaging involves the inversion of a diffraction pattern to find the wave function at the exit-surface plane of the specimen. It is a promising technique for imaging, for example, nanoparticles with electrons and biological molecules with X-rays. If the illumination is not a plane wave of infinite extent, then a relative drift between the illumination and the object introduces errors into the diffraction pattern; an issue which is often overlooked. This may be of particular importance for applications with electron microscopes which use nanoscale probes. Here we show that beams which are uniform over a sufficiently large region can be used to pose a phase retrieval problem that is immune from specimen drift, provided suitable analysis of the diffraction data is undertaken. The method only applies to objects contained within a support that is smaller than a uniform region of the beam.     

A method of combining STEM image with parallel beam diffraction and electron-optical conditions for diffractive imaging

We describe a method of combining STEM imaging functionalities with nanoarea parallel beam electron diffraction on a modern TEM. This facilitates the search for individual particles whose diffraction patterns are needed for diffractive imaging or structural studies of nanoparticles. This also lays out a base for 3D diffraction data collection.     

Real Time TEM Imaging of Compression and Shear of Single Fullerene-Like MoS2 Nanoparticle

The deformation and degradation behavior of single inorganic fullerenes nanoparticles of MoS₂ under compression and shear has been observed in real time using a high-resolution transmission electron microscope equipped with a nanoindentation holder. The MoS₂ nanoparticles were compressed using a nanoindenter and a truncated diamond tip. For the first time, real time imaging of the deformation of individual nanoparticles clearly shows first orientation changes in the particle shape during loading process followed by a large deformation and the exfoliation of the outer sheets of the fullerene nested structure. Exfoliation was observed for a contact pressure estimated at 1 GPa. Additional sliding tests performed with the nanoindenter gave evidence for a rolling process for lower contact pressures up to 100 MPa.