Techniques for Enhancing Contrast In the Electron Microscopy of Biological Specimens

Recent developments in methods for enhancing contrast in the electron microscopy of biological specimens are reviewed. The treatment of tissue sections with various reagents and possible histochemical methods applicable to electron microscopy are discussed. For the study of viruses, bacteriophages and the larger protein molecules high resolution can be achieved in the negative contrast technique.     

Electron Holography of Organic and Biological Materials

Electron holography offers the advantage of high resolution imaging of electric and magnetic fields, which is rarely achieved by other microscopic techniques. The variety of scientific tasks investigated by electron holography extends from subatomic‐resolution imaging of inorganic crystal structures up to morphological studies of polymers or biological samples by making use of subtle differences in their chemical composition or density. In this paper, we discuss basic prospects and applications of holography in the field of organic semiconductor devices and biological samples, as well as in polymer science.     

Visual Contrast In Photographic Prints*

A method of predicting visual contrast in the context of a photographic print is described. The method employs an equation derived from functions of the density scale, average gradient and density frequency response of the objective tone reproduction. Predictions of visual contrast based on this method are in good agreement with average evaluations of contrast made by a panel of 20 observers on a variety of photographic prints. The method also provides information on the relative contributions to over-all contrast of the factors in the prediction equation. In addition, the results provide an indication of the threshold for detection of over-all contrast in the context of a photographic reproduction.     

Biological and Magnetic Contrast Evaluation of Shape-Selective Mn–Fe Nanowires

One-dimensional composite Mn–Fe oxide nanostructures of different sizes (nanoneedles, nanorods, and nanowires) were prepared by a linker-induced organization of manganese-doped iron oxide nanoparticles. The nanostructures were obtained by the treatment of $hbox{MnFe}_{2}hbox{O}_{4}$ nanoparticles in the presence of cystamine. The average lengths of nanoneedle, nanorod, and nanowire are approximately 400, 800, and 1000 nm, respectively. High-resolution transmission electron microscopy (HR-TEM), energy-dispersive X-ray (EDX) spectroscopy, and inductively coupled plasma-optical emission spectroscopy (ICP–OES) were employed to characterize the morphologies and the elemental contents of the nanostructures. As an example of their potential applications, these nanostructures were explored as the cell-labeling agents for magnetic resonance imaging (MRI). The magnetic contrast properties of the nanostructures were characterized by a 1.5 T (Tesla) whole body MR system. 10 $mu,$g/mL of the nanostructures caused substantial negative contrast. After in vitro incubation, the nanostructures could be effectively incorporated into the cells of a monocyte/macrophage cell line (RAW264.7). These cells’ viability and proliferation potential were not affected when the labeling concentration was less than 50 $mu,$g/mL.      

Application of Ciné Techniques In Electron Microscopy

Ciné camera recordings have been made of (a) the movement of dislocations in thin metal foils, (b) contrast changes in single crystals of colloidal silver iodide. Electron diffraction pattern changes have also been recorded on ciné film. Methods and apparatus are discussed together with recent attempts to film structural details in biological objects examined in the electron microscope.     

锗硅材料在微电子、光电子方面的应用

锗硅材料的制造与硅平面工艺相容，且具有优良的电学、光学性能，被称为“第二代硅微电子技术”。在高速异质结双极型晶体管（ＨＢＴ）、调制掺杂场效管中获得应用，其中ＨＢＴ即将走向产业化，锗硅材料具有能带可调的特性，在光电子方面有其应用价值，锗硅材料做红外探测器有诱人的前景。     

A Functional CT Contrast Agent for In Vivo Imaging of Tumor Hypoxia

Hypoxia, which has been well established as a key feature of the tumor microenvironment, significantly influences tumor behavior and treatment response. Therefore, imaging for tumor hypoxia in vivo is warranted. Although some imaging modalities for detecting tumor hypoxia have been developed, such as magnetic resonance imaging, positron emission tomography, and optical imaging, these technologies still have their own specific limitations. As computed tomography (CT) is one of the most useful imaging tools in terms of availability, efficiency, and convenience, the feasibility of using a hypoxia‐sensitive nanoprobe (Au@BSA‐NHA) for CT imaging of tumor hypoxia is investigated, with emphasis on identifying different levels of hypoxia in two xenografts. The nanoprobe is composed of Au nanoparticles and nitroimidazole moiety which can be electively reduced by nitroreductase under hypoxic condition. In vitro, Au@BSA‐NHA attain the higher cellular uptake under hypoxic condition. Attractively, after in vivo administration, Au@BSA‐NHA can not only monitor the tumor hypoxic environment with CT enhancement but also detect the hypoxic status by the degree of enhancement in two xenograft tumors with different hypoxic levels. The results demonstrate that Au@BSA‐NHA may potentially be used as a sensitive CT imaging agent for detecting tumor hypoxia.     

The In Vivo Radiosensitizing Effect of Gold Nanoparticles Based MRI Contrast Agents

Owing to the high atomic number (Z) of gold element, the gold nanoparticles appear as very promising radiosensitizing agents. This character can be exploited for improving the selectivity of radiotherapy. However, such an improvement is possible only if irradiation is performed when the gold content is high in the tumor and low in the surrounding healthy tissue. As a result, the beneficial action of irradiation (the eradication of the tumor) should occur while the deleterious side effects of radiotherapy should be limited by sparing the healthy tissue. The location of the radiosensitizers is therefore required to initiate the radiotherapy. Designing gold nanoparticles for monitoring their distribution by magnetic resonance imaging (MRI) is an asset due to the high resolution of MRI which permits the accurate location of particles and therefore the determination of the optimal time for the irradiation. We recently demonstrated that ultrasmall gold nanoparticles coated by gadolinium chelates (Au@DTDTPA‐Gd) can be followed up by MRI after intravenous injection. Herein, Au@DTDTPA and Au@DTDTPA‐Gd were prepared in order to evaluate their potential for radiosensitization. Comet assays and in vivo experiments suggest that these particles appear well suited for improving the selectivity of the radiotherapy. The dose which is used for inducing similar levels of DNA alteration is divided by two when cells are incubated with the gold nanoparticles prior to the irradiation. Moreover, the increase in the lifespan of tumor bearing rats is more important when the irradiation is performed after the injection of the gold nanoparticles. In the case of treatment of rats with a brain tumor (9L gliosarcoma, a radio‐resistant tumor in a radiosensitive organ), the delay between the intravenous injection and the irradiation was determined by MRI.     

Thermal Conductivity Measurement of Anisotropic Biological Tissue In Vitro

The accurate determination of the thermal conductivity of biological tissues has implications on the success of cryosurgical/hyperthermia treatments. In light of the evident anisotropy in some biological tissues, a new modified stepwise transient method was proposed to simultaneously measure the transverse and longitudinal thermal conductivities of anisotropic biological tissues. The physical and mathematical models were established, and the analytical solution was derived. Sensitivity analysis and experimental simulation were performed to determine the feasibility and measurement accuracy of simultaneously measuring the transverse and longitudinal thermal conductivities. The experimental system was set up, and its measurement accuracy was verified by measuring the thermal conductivity of a reference standard material. The thermal conductivities of the pork tenderloin and bovine muscles were measured using the traditional 1D and proposed methods, respectively, at different temperatures. Results indicate that the thermal conductivities of the bovine muscle are lower than those of the pork tenderloin muscle, whereas the bovine muscle was determined to exhibit stronger anisotropy than the pork tenderloin muscle. Moreover, the longitudinal thermal conductivity is larger than the transverse thermal conductivity for the two tissues and all thermal conductivities increase with the increase in temperature. Compared with the traditional 1D method, results obtained by the proposed method are slightly higher although the relative deviation is below 5 %.     

Cyclic Saturation Dislocation Pattern Observation in the Vicinity of Grain Boundaries by Electron Channeling Contrast Technique

State Key Laboratory for Fatigue and Fracture of Materials, Institute of Metal Research, Chinese Academy of Sciences,Shenyang, 110015, China)Abstract:The cyclic saturation dislocation patterns within grains and in the vicinity of low-angle grain boundaries in fatigued copper crystal were successfully observed by electron channeling contrast technique in SEM. The results show that the dislocation patterns within grains consisted of typical two-phase structure, i.e. persistent slip bands (PSB) and veins. With increasing plastic strain amplitude (γp1 ≥1.7×10-3), large amount of PSBs and regufar dislocation walls were observed.The dislocation walls and PSBs could cross through the low-angle grain boundaries continuously except that the dislocation-free zone (DFZs) appeared at some local regions. Combining with the cyclic stress-strain response and dislocation patterns, the effect of low-angle grain boundaries on cyclic deformation behavior was discussed.     

Recent Progress of In Situ Transmission Electron Microscopy for Energy Materials

In situ transmission electron microscopy (TEM) is one of the most powerful approaches for revealing physical and chemical process dynamics at atomic resolutions. The most recent developments for in situ TEM techniques are summarized; in particular, how they enable visualization of various events, measure properties, and solve problems in the field of energy by revealing detailed mechanisms at the nanoscale. Related applications include rechargeable batteries such as Li-ion, Na-ion, Li–O₂, Na–O₂, Li–S, etc., fuel cells, thermoelectrics, photovoltaics, and photocatalysis. To promote various applications, the methods of introducing the in situ stimuli of heating, cooling, electrical biasing, light illumination, and liquid and gas environments are discussed. The progress of recent in situ TEM in energy applications should inspire future research on new energy materials in diverse energy-related areas.     

In Situ Transmission Electron Microscopy for Energy Materials and Devices

Energy devices such as rechargeable batteries, fuel cells, and solar cells are central to powering a renewable, mobile, and electrified future. To advance these devices requires a fundamental understanding of the complex chemical reactions, material transformations, and charge flow that are associated with energy conversion processes. Analytical in situ transmission electron microscopy (TEM) offers a powerful tool for directly visualizing these complex processes at the atomic scale in real time and in operando. Recent advancements in energy materials and devices that have been enabled by in situ TEM are reviewed. First, the evolutionary development of TEM nanocells from the open‐cell configuration to the closed‐cell, and finally the full‐cell, is reviewed. Next, in situ TEM studies of rechargeable ion batteries in a practical operation environment are explored, followed by applications of in situ TEM for direct observation of electrocatalyst formation, evolution, and degradation in proton‐exchange membrane fuel cells, and fundamental investigations of new energy materials such as perovskites for solar cells. Finally, recent advances in the use of environmental TEM and cryogenic electron microscopy in probing clean‐energy materials are presented and emerging opportunities and challenges in in situ TEM research of energy materials and devices are discussed.     

In Situ Repair of 2D Chalcogenides under Electron Beam Irradiation

Molybdenum disulfide (MoS₂) and bismuth telluride (Bi₂Te₃) are the two most common types of structures adopted by 2D chalcogenides. In view of their unique physical properties and structure, 2D chalcogenides have potential applications in various fields. However, the excellent properties of these 2D crystals depend critically on their crystal structures, where defects, cracks, holes, or even greater damage can be inevitably introduced during the preparation and transferring processes. Such defects adversely impact the performance of devices made from 2D chalcogenides and, hence, it is important to develop ways to intuitively and precisely repair these 2D crystals on the atomic scale, so as to realize high‐reliability devices from these structures. Here, an in situ study of the repair of the nanopores in MoS₂ and Bi₂Te₃ is carried out under electron beam irradiation by transmission electron microscopy. The experimental conditions allow visualization of the structural dynamics of MoS₂ and Bi₂Te₃ crystals with unprecedented resolution. Real‐time observation of the healing of defects at atomic resolution can potentially help to reproducibly fabricate and simultaneously image single‐crystalline free‐standing 2D chalcogenides. Thus, these findings demonstrate the viability of using an electron beam as an effective tool to precisely engineer materials to suit desired applications in the future.     

Tuning the Surface Electron Accumulation Layer of In2O3 by Adsorption of Molecular Electron Donors and Acceptors

In₂O₃, an n-type semiconducting transparent transition metal oxide, possesses a surface electron accumulation layer (SEAL) resulting from downward surface band bending due to the presence of ubiquitous oxygen vacancies. Upon annealing In₂O₃ in ultrahigh vacuum or in the presence of oxygen, the SEAL can be enhanced or depleted, as governed by the resulting density of oxygen vacancies at the surface. In this work, an alternative route to tune the SEAL by adsorption of strong molecular electron donors (specifically here ruthenium pentamethylcyclopentadienyl mesitylene dimer, [RuCp*mes]₂) and acceptors (here 2,2′-(1,3,4,5,7,8-hexafluoro-2,6-naphthalene-diylidene)bis-propanedinitrile, F₆TCNNQ) is demonstrated. Starting from an electron-depleted In₂O₃ surface after annealing in oxygen, the deposition of [RuCp*mes]₂ restores the accumulation layer as a result of electron transfer from the donor molecules to In₂O₃, as evidenced by the observation of (partially) filled conduction sub-bands near the Fermi level via angle-resolved photoemission spectroscopy, indicating the formation of a 2D electron gas due to the SEAL. In contrast, when F₆TCNNQ is deposited on a surface annealed without oxygen, the electron accumulation layer vanishes and an upward band bending is generated at the In₂O₃ surface due to electron depletion by the acceptor molecules. Hence, further opportunities to expand the application of In₂O₃ in electronic devices are revealed.     

Fuzzy Reasoning to More Accurately Determine Void Areas on Optical Micrographs of Composite Structures

Accomplishing the best-performing composite matrix (resin) requires that not only the processing method but also the cure cycle generate low-void-content structures. If voids are present, the performance of the composite matrix will be significantly reduced. This is usually noticed by significant reductions in matrix-dominated properties, such as compression and shear strength. Voids in composite materials are areas that are absent of the composite components: matrix and fibers. The characteristics of the voids and their accurate estimation are critical to determine for high performance composite structures. One widely used method of performing void analysis on a composite structure sample is acquiring optical micrographs or Scanning Electron Microscope (SEM) images of lateral sides of the sample and retrieving the void areas within the micrographs/images using an image analysis technique. Segmentation for the retrieval and subsequent computation of void areas within the micrographs/images is challenging as the gray-scaled values of the void areas are close to the gray-scaled values of the matrix leading to the need of manually performing the segmentation based on the histogram of the micrographs/images to retrieve the void areas. The use of an algorithm developed by NASA and based on Fuzzy Reasoning (FR) proved to overcome the difficulty of suitably differentiate void and matrix image areas with similar gray-scaled values leading not only to a more accurate estimation of void areas on composite matrix micrographs but also to a faster void analysis process as the algorithm is fully autonomous.     

Rational In Silico Design of an Organic Semiconductor with Improved Electron Mobility

Organic semiconductors find a wide range of applications, such as in organic light emitting diodes, organic solar cells, and organic field effect transistors. One of their most striking disadvantages in comparison to crystalline inorganic semiconductors is their low charge‐carrier mobility, which manifests itself in major device constraints such as limited photoactive layer thicknesses. Trial‐and‐error attempts to increase charge‐carrier mobility are impeded by the complex interplay of the molecular and electronic structure of the material with its morphology. Here, the viability of a multiscale simulation approach to rationally design materials with improved electron mobility is demonstrated. Starting from one of the most widely used electron conducting materials (Alq₃), novel organic semiconductors with tailored electronic properties are designed for which an improvement of the electron mobility by three orders of magnitude is predicted and experimentally confirmed.