A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.     

Nanoscratch study of Zn1−xMnxO heteroepitaxial layers

We investigated the nanotribological properties of Zn_1−xMn_xO epilayers (0 ≤ x ≤ 0.16) grown by molecular beam epitaxy (MBE) on sapphire substrates. The surface roughness and friction coefficient (μ) were analyzed by means of atomic force microscopy (AFM) and hysitron triboscope nanoindenter techniques.The nanoscratch system gave the μ value of the films ranging from 0.17 to 0.07 and the penetration depth value ranging 294-200 nm when the Mn content was increased from x = 0 to 0.16. The results strongly indicate that the scratch wear depth under constant load shows that higher Mn content leads to Zn_1−xMn_xO epilayers with higher shear resistance, which enhances the Mn-O bond. These findings reveal that the role of Mn content on the growth of Zn_1−xMn_xO epilayers can be identified by their nanotribological behavior.     

Nanoscratch behavior of Zn1−xCdxSe heteroepitaxial layers

This paper describes the nanoscratch behavior of Zn_1−xCd_xSe epilayers grown using molecular beam epitaxy (MBE). Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Hysitron Triboscope nanoindenter techniques were employed to determine the microstructures, morphologies, friction coefficients (μ), and hardnesses (H) of these materials, and thereby propose an explanation for their properties in terms of nanotribological behavior. Nanoscratch analysis revealed that the coefficient of friction of the Zn_1−xCd_xSe epilayer system decreased from 0.172 to 0.139 upon increasing the Cd content (x) from 0.07 to 0.34. Furthermore, studies of the scratch wear depth under a ramping load indicated that a higher Cd content provided the Zn_1−xCd_xSe epilayers with a higher shear resistance, which enhanced the strength of the CdSe bonds. These findings suggest that the greater stiffness of the CdSe bond, relative to that of the ZnSe bond, enhances the hardness of the epilayers. Indeed, the effect of the Cd content on the growth of the Zn_1−xCd_xSe epilayers is manifested in the resulting nanotribological behavior.     

A method of correlative light and electron microscopy for yeast cells

Correlative light and electron microscopy (CLEM) is a method of imaging in which the same specimen is observed by both light microscopy and electron microscopy. Specifically, CLEM compares images obtained by light and electron microscopy and makes a correlation between them. After the advent of fluorescent proteins, CLEM was extended by combining electron microscopy with fluorescence microscopy to enable molecular-specific imaging of subcellular structures with a resolution at the nanometer level. This method is a powerful tool that is used to determine the localization of specific molecules of interest in the context of subcellular structures. Knowledge of the localization of target proteins coupled with the functions of the structures to which they are localized yields valuable information about the molecular functions of these proteins. However, this method has been mostly applied to adherent cells due to technical difficulties in immobilizing non-adherent target cells, such as yeasts, during sample preparation. We have developed a method of CLEM applicable to yeast cells. In this report, we detail this method and present its extension to Live CLEM. The Live CLEM method enabled us to link the dynamic properties of molecules of interest to cellular ultrastructures in the yeast cell. Since yeasts are premier organisms in molecular genetics, combining CLEM with yeast genetics promises to provide important new findings for understanding the molecular basis of the function of cellular structures.     

Ultrastructure of antennal and posterior abdominal sensilla in Chlorophorus caragana females

Chlorophorus caragana Xie & Wang is a destructive wood-boring beetle that damages Caragana spp. bushes and is distributed in desert areas in north-west China. Using scanning electron microscopy and transmission electron microscopy, we observed the morphology and ultrastructure of antennal and posterior abdominal sensilla in C. caragana females, to discuss the putative functions of these sensilla in host location and oviposition behaviors.In total, seven types (24 subtypes) of sensilla were located on the antenna and posterior abdomen. On the antenna, there were Böhm's bristles (BB.); four subtypes of sensilla chaetica (Ch.1–Ch.4) characterized by non-porous surfaces and sensillum-lymph cavities without dendrites; two subtypes of sensilla trichodea (Tr.1 and Tr.2) with a tip pore and dendrites surrounded by dendritic sheaths; dome-shaped sensilla (Dom.) emerging from a deep cavity with one tip pore; four subtypes of sensilla basiconica (Ba.1–Ba.4) and one type of sensilla auricillica (Au.) with a porous cuticular surface and dendrites in the sensillum-lymph cavity; and one type of sensilla styloconica (Sty.) with grooves on the cuticular wall. On the posterior abdomen, there were four subtypes of sensilla chaetica (Ch.5–Ch.8); three subtypes of sensilla trichodea (Tr.3–Tr.5); and three subtypes of sensilla basiconica (Ba.5–Ba.7; Ba.5 had no groove in the cuticular wall, Ba.6 had one tip pore, and Ba.7 was located in a cuticular cavity). The antennal sensilla were believed to be mechanosensitive, chemosensitive, and sensitive to humidity and temperature, and to play roles in mating, host location and oviposition. The abdominal sensilla are believed to be related to oviposition behaviors.     

Antenna morphology and sensilla ultrastructure of Tetrigus lewisi Candèze (Coleoptera: Elateridae)

We used scanning and transmission electron microscopy to study the typology, morphology, distributions, and ultrastructures of the antennal sensilla of Tetrigus lewisi Candèze, a predatory click beetle that feeds on longhorned beetles, such as, Monochamus alternatus (Coleoptera: Cerambycidae). We observed eight types of sensilla on the antennae, including sensilla chaetica (with three subtypes: ch.1, ch.2, ch.3), sensilla basiconica (subtypes: ba.1, ba.2, ba.3), sensilla trichodea (subtypes: tr.1, tr.2), as well as sensilla auricillica, sensilla coeloconica, sensilla campaniformia, sensilla styloconica and Böhm's bristles. Significant sexual dimorphism was found in the antenna morphology, as well as in the density of type 2 sensilla trichodea and type 1 sensilla basiconica. We observed thick cuticular walls on sensilla chaetica, sensilla trichodea and sensilla campaniformia; clear pore structures on sensilla trichodea, sensilla basiconica and sensilla auricillica; and double walls with spoke-channels on sensilla coeloconica. The chemoreception, mechanoreception and thermo-/hygro-reception functions were deduced from fine structures on the cuticular walls and the dendrites of the different sensilla types. We suggest that all these sensilla have important roles in the host location, mating and predatory behavior of T. lewisi.     

Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections

In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions.     

The Microstructure of Soda-Lime Silicate Glasses Detected by Electron Microscopy

Transmission electron microscopy and selected area electron diffraction performances were used to characterise the microstructure of a multicomponent soda-lime silicate glass, the composition of which corresponds to the miscibility gap in the SiO₂—Na₂O system. It has been stated that phase separation already exists in as prepared specimens, and the Na₂O-rich phase occurs in the form of nearly spherical droplets. The microstructure of these glasses was modified by high-temperature annealing in air and hydrogen, by irradiation with soft X- and gamma-irradiation and by mechanical stretching. In this way the quantity, size, shape and distribution of these particles was changed. A variety of induced changes affects the structure-sensitive properties of commercial soda lime silicate glasses, which can be potentially useful for new applications.     

Nano-cracks in a synthetic graphite composite for nuclear applications

Mrozowski nano-cracks in nuclear graphite were studied by transmission electron microscopy and selected area diffraction. The material consisted of single crystal platelets typically 1–2 nm thick and stacked with large relative rotations around the c-axis; individual platelets had both hexagonal and cubic stacking order. The lattice spacing of the (0002) planes was about 3% larger at the platelet boundaries which were the source of a high fraction of the nano-cracks. Tilting experiments demonstrated that these cracks were empty, and not, as often suggested, filled by amorphous material. In addition to conventional Mrozowski cracks, a new type of nano-crack is reported, which originates from the termination of a graphite platelet due to crystallographic requirements. Both types are crucial to understanding the evolution of macro-scale graphite properties with neutron irradiation.     

Ultrasmall Ge islands with low diameter-to-height aspect ratio on Si(1 0 0)-(2 × 1) surfaces

Scanning tunneling microscopy (STM) and high resolution cross-sectional transmission electron microscopy (XTEM) studies have been used to investigate the formation of Ge nanocrystals grown on Si(1 0 0)-(2 × 1) surfaces by molecular beam epitaxy (MBE). We observe relatively high density of Ge islands where small ‘pyramids’, small ‘domes’ and facetted ‘domes’ of various sizes co-exist in the film. As revealed from XTEM images, a large fraction of islands, especially dome-shaped Ge islands have been found to have an aspect ratio of ∼1 (diameter):1 (height). Observation of truncated-sphere-shaped Ge islands with a narrow neck contact with the wetting layer is reported.     

Field emission characteristics of carbon nanotubes post-treated with high-density Ar plasma

The field emission characteristics of carbon nanotubes (CNTs) grown by thermal chemical vapor deposition (CVD) and subsequently surface treated by high-density Ar plasma in an inductively coupled plasma reactive ion etching (ICP-RIE) with the various plasma powers were measured. Results indicate that, after treated by Ar plasma with power between 250 and 500 W, the emission current density of the CNTs is enhanced by nearly two orders of magnitude (increased from 0.65 to 48 mA/cm²) as compared to that of the as-grown ones. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to investigate the structural features relevant to the modified field emission properties of CNTs. The SEM images of CNTs subjected to a 500 W Ar plasma treatment exhibit obvious damages to the CNTs. Nevertheless, the turn-on fields decreased from 3.6 to 2.2 V/μm, indicating a remarkable field emission enhancement. Our results further suggest that the primary effect of Ar plasma treatment might be to modify the geometrical structures of the local emission region in CNTs. In any case, the Ar plasma treatment appears to be an efficient method to enhance the site density for electron emission and, hence markedly improving the electric characteristics of the CNTs.     

Micro- and nano-structural analyses of damage in bone

Skeletal fractures represent a significant medical and economic burden for our society. In the US alone, age-related hip, spine, and wrist fractures accounted for more than $17 billion in direct health care costs in 2001. Moreover, skeletal fractures are not limited to the elderly; stress fractures and impact/trauma-related fractures are a significant problem in younger people also. Gaining insight into the mechanisms of fracture and how these mechanisms are modulated by intrinsic as well as extrinsic factors may improve the ability to define fracture risk and develop and assess preventative therapies for skeletal fractures. Insight into failure mechanisms of bone, particularly at the ultrastructural-level, is facilitated by the development of improved means of defining and measuring tissue quality. Included in these means are microscopic and spectroscopic techniques for the direct observation of crack initiation, crack propagation, and fracture behavior. In this review, we discuss microscopic and spectroscopic techniques, including laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopic imaging for visually observing microdamage in bone, and the current understanding of damage mechanisms derived from these techniques.     

飞秒时间分辨的能谱分析光电子显微镜：异质结超快载流子动力学测量

本文成功搭建了一套集成了能谱分析功能的时间分辨光电子显微镜系统(TR-PEEM),能够对电子密度分布进行时间分辨和能量分辨的成像.这套4D显微镜在空间、时间、能量多维度获取电子动力学信息提供了前所未有的手段.本文使用184 fs的时间分辨、150 meV的能量分辨和优于150 nm的空间分辨对半导体进行了测量,在Si(111)表面的Pb岛上获得了微区光电子能谱和能量分辨的TR-PEEM图像.实验结果表明,这套系统是进行异质结载流子动力学观察的有力工具,有助于在亚微米/纳米空间尺度和超快时间尺度上加深对半导体性质的理解.     

Modern contaminants affecting microscopic residue analysis on stone tools: A word of caution

Residue analysis is a method frequently used to infer the function of stone tools and it is very often applied in combination with use-wear analysis. Beyond its undeniable potential, the method itself has several intrinsic constraints. Apart from the exceptional circumstances necessary for residues to survive, the correct identification of the residue type is a very debatable topic. Before attempting to recognise ancient residues, a proper method should allow analysts to identify possible modern contaminants and exclude them from the final interpretation. Therefore, analysts should not underestimate the presence of modern contaminants and might learn how to discriminate the background noise due to handling.The main aim of this research is to provide some methodological improvements to residue analysis through the characterisation of some modern residues often present on the surface of stone tools (e.g. skin flakes, modelling clay). This characterisation was done by using both optical light microscopy (OLM) and scanning electron microscopy (SEM).Finally, a special care in the post-excavation treatment of stone tools is claimed in order to avoid major contamination of the samples.     

Ultrastructure of antennal sensilla of an autoparasitoid Encarsia sophia (Hymenoptera: Aphelinidae)

Encarsia sophia (Hymenoptera: Aphelinidae) is a parasitoid utilized for biological control of Bemisia tabaci, with selection of prey aided by chemoreceptor organs. The morphology and distribution of the antennal sensilla (chemoreceptors) of E. sophia were examined using Transmission electron micrographs. The total antennal length for E. sophia was 429.28 ± 0.95 μm for females and 437.19 ± 8.21 for males, and each antennae was found to consist of seven sensilla of different types. Both sexes possessed sensilla chaetica, sensilla trichodea, basiconic capitate peg sensilla, multiporous grooved-surface placoid sensilla (MG-PS), uniporous rod-like sensilla, nonporous finger-like sensilla, and sensilla coeloconica. Transmission electron micrographs of longitudinal sections of female antennae showed that they were composed of fat body, cuticle, mesoscutello-metanotal muscles, neurons, and glandular tissue, and cross-sections of the basal MG-PS showed sensillar lymph cavities and dendrites. The MG-PSs were imbedded in an electron-dense mass with cuticular invaginations which acted as pores that connected to a central lumen. The possible function of each type of sensilla is discussed.     

低能电子显微术及其应用

低能电子显微术是新发展起来的一种显微探测技术。它的特点是利用低能（１－３０ｅＶ）电子的弹性背散射使表面实空间实时成像，具有高的横向（１５ｎｍ）和纵向（原子级）分辩率，且易与低能电子衍射及其他电子显微术相结合。近年来它已有效地应用于金属和半导体表面的形貌观测、表面相变、吸附、反应及生长过程的研究。     

Metal–Polymer Hybrid Nanoparticles for Correlative High‐Resolution Light and Electron Microscopy

The combination of fluorescence microscopy and electron microscopy promises a deeper insight into the ultrastructural features of cell organelles, e.g., after drug administration. Both methods complement each other and provide, as a correlative approach, a keen insight into the fate of nanoparticles within the cell. Moreover, it represents a promising tool to determine alterations of the cellular environment as a response to particle uptake. However, the availability of suitable correlative markers is mandatory for such correlative approaches. In this contribution, the utilization of poly(ethylene imine) based metal–polymer hybrid particles labeled with small gold nanoparticles and Rhodamine B facilitating the observation of the particles by means of fluorescence as well as by transmission electron microscopy is suggested. Correlative light and electron microscopy is used to study uptake and intracellular fusion processes of endosomal/lysosomal structures.