Alexandra E. Porter

Direct imaging of single-walled carbon nanotubes in cells

The development of single-walled carbon nanotubes for various biomedical applications is an area of great promise. However, the contradictory data on the toxic effects of single-walled carbon nanotubes highlight the need for alternative ways to study their uptake and cytotoxic effects in cells. Single-walled carbon nanotubes have been shown to be acutely toxic in a number of types of cells, but the direct observation of cellular uptake of single-walled carbon nanotubes has not been demonstrated previously due to difficulties in discriminating carbon-based nanotubes from carbon-rich cell structures. Here we use transmission electron microscopy and confocal microscopy to image the translocation of single-walled carbon nanotubes into cells in both stained and unstained human cells. The nanotubes were seen to enter the cytoplasm and localize within the cell nucleus, causing cell mortality in a dose-dependent manner.

Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics

The incorporation of silicate into hydroxyapatite (HA) has been shown to significantly increase the rate of bone apposition to HA bioceramic implants. However, uncertainty remains about the mechanism by which silicate increases the in vivo bioactivity of HA. In this study, high-resolution transmission electron microscopy was used to observe dissolution from HA, 0.8 wt% Si-HA and 1.5 wt% Si-HA implants after 6 and 12 weeks in vivo. Our observations confirmed that defects, in particular those involving grain boundaries, were the starting point of dissolution in vivo. Dissolution was observed to follow the order 1.5 wt% Si-HA>0.8 wt% Si-HA>pure HA and it was found to be particularly prevalent at grain boundaries and triple-junctions. These observations may help to explain the mechanism by which silicate ions increase the in vivo bioactivity of pure HA, and highlight the enhanced potential of these ceramics for biomedical applications.

The recrystallization of nickel-base superalloys

The effects of recrystallization on the γ′ distribution in four nickel-base superalloys of varying γ′ volume fraction (Nimonics PE16, 80A and 115, and Udimet 720) have been studied by transmission electron microscopy. These effects are explained in terms of high solubility and diffusivity in the recrystallization interface, and it is suggested that high diffusivity assumes greater importance as the amount of solute dissolved in the boundary increases. Some attention is given to the nucleation of recrystallization. It is shown that in one of the alloys (Udimet 720), nucleation at grain boundaries involves subgrain coalescence. Subsequent growth of the nucleus occurs by strain-induced boundary migration.

Imaging carbon nanoparticles and related cytotoxicity

Carbon-based nanoparticles have attracted significant attention due to their unique physical, chemical, and electrical properties. Numerous studies have been published on carbon nanoparticle toxicity; however, the results remain contradictory. An ideal approach is to combine a cell viability assay with nanometer scale imaging to elucidate the detailed physiological and structural effects of cellular exposure to nanoparticles. We have developed and applied a combination of advanced microscopy techniques to image carbon nanoparticles within cells. Specifically, we have used EFTEM, HAADF-STEM, and tomography and confocal microscopy to generate 3-D images enabling determination of nanoparticle spatial distribution in a cell. With these techniques, we can differentiate between the carbon nanoparticles and the cell in both stained and unstained sections. We found carbon nanoparticles (C60, single-walled carbon nanotubes (SWNT), and multi-walled carbon nanotubes (MWNT)) within the cytoplasm, lysosomes, and nucleus of human monocyte-derived macrophage cells (HMM). C60 aggregated along the plasma and nuclear membrane while MWNTs and SWNTs were seen penetrating the plasma and nuclear membranes. Both the Neutral Red (NR) assay and ultra-stuctural analysis showed an increase in cell death after exposure to MWNTs and SWNTs. SWNTs were more toxic than MWNTs. For both MWNTs and SWNTs, we correlated uptake of the nanoparticles with a significant increase in necrosis. In conclusion, high resolution imaging studies provide us with significant insight into the localised interactions between carbon nanoparticles and cells. Viability assays alone only provide a broad toxicological picture of nanoparticle effects on cells whereas the high resolution images associate the spatial distributions of the nanoparticles within the cell with increased incidence of necrosis. This combined approach will enable us to probe the mechanisms of particle uptake and subsequent chemical changes within the cell, essential for identifying the toxicological profiles of carbon nanoparticles.

Ultrastructural Characterisation of Hydroxyapatite and Silicon-Substituted Hydroxyapatite

A recent in vivo study comparing bone apposition to hydroxyapatite (HA) and silicon substituted HA (Si-HA) ceramic implants demonstrated that bone app osition was significantly increased at the surface of Si-HA ceramics. However, the me chanism by which silicon increases the in vitro and in vivo bioactivity is still unresolved. In this study, defect structures in phase pure HA and Si-HA were observed and characterized for the first time us ing high-resolution transmission microscopy (HR-TEM). The results suggest that an increased number of d fect structures in Si-HA may be playing a significant role in increasing the solubility of HA and the subsequent rate at which bone apposes HA ceramics.

Syntheses of Silicon-Containing Apatite Fibres by a Homogeneous Precipitation Method and Their Characterization

Silicon-containing apatite (Si-HAp) fibres were successfully synthesized by a homogeneous precipitation method. The resulting Si-HAp fibres were composed of carbonate-containing apatite fibres with preferred orientation in the c-axis. The Si contents in the Si-HAp fibres could be controlled by the Si concentration of the starting solutions. TEM observation indicated that the Si-HAp fibres were of single crystal. The Si-HAp fibres have potential as novel materials for high-performance biomedical devices.

High Resolution Transmission Electron Microscopy Investigation of Single Crystal Apatite Fibres Synthesized by using a Homogeneous Precipitation Method

Apatite fibres were synthesized from aqueous solutions containing ur ea in the Ca(NO3)2-(NH4)2HPO4-HNO3 systems by a homogeneous precipitation method. The products were composed of carbonate-containing apatite fibres with preferred orienta tion along the (h00) planes. In order to characterise the properties of individual fibres, an investig ation was performed using a high-resolution transmission electron microscopy (HR-TEM). It was confirmed, from the results of HR-TEM, that the apatite fibres were single crystals with the c-axis orientation parallel to the long axis of the fibre. In addition, the fibres were highly strained defects and consisted of domains that were preferentially oriented with the a-axis parallel to the surface of the substr ate.

Microstructural Changes of Single-Crystal Apatite Fibres during Heat Treatment

The apatite fibres were prepared using a homogeneous precipitati on route. The as-prepared fibres were composed of carbonate-containing apatite wi th preferred orientation along the c-axis direction. The fibres were highly strained and were composed of domains that preferentially oriented themselves with the c-axis parallel to the sur face of the substrate. When the as-synthesized fibres were heated at 800 ̊C to 1200 ̊C for 1 h, the domain structure of the fibres disappeared but they remained highly strained. In the cases of the apatite fibres heated at 1000 ̊C and 1200 ̊C, their microstructures changed during sintering to form some grain boundaries and dislocations. The apatite fibres heated at 1200 ̊C for 1 h contained ma ny angular voids formed by releasing the carbonate groups during sintering. Introduction Hydroxyapatite (HAp) has widely been applied as a biomaterial for substituting human hard tissues. We have successfully synthesized single-crystal apatite fi br s by a homogeneous precipitation method [1] and then characterized specific properties of the apatit e fibres by high-resolution transmission electron microscopy (HR-TEM) [2]. The as-synthesi zed apatite fibres were composed of carbonate-containing apatite with a long-axis dimension of 60-100 μm. The fibres have a preferred orientation in the c-axis direction and therefor developed the a-plane of the HAp crystal. In addition, the fibres were highly strained and were com posed of domains that preferentially oriented themselves with the c-axis parallel to the surface of the substrate. A sintering process is needed to fabricate high-performance bioma terials, such as porous ceramics [1], bioactive HAp/polymer hybrids [3, 4] and scaffolds for tissue engineer i g of bone [5, 6], from our current apatite fibres. Therefore, it is important to elucidat e how the microstructure of the apatite fibres changes during heat treatment. The objectives of this i nvest gation were to examine the microstructure of the apatite fibres heated at 800 ̊C, 1000 ̊C and 1200 ̊C for 1 h using several characterization techniques and to compare these microstructures to those of apatite fibres that had not been heat-treated. Materials and Methods Apatite fibres were prepared by the same process as given i n Ref [1]. The resulting apatite-fibre sheets were heated at 800 ̊C, 1000 ̊C and 1200 ̊C for 1 h at a ramp-rate of 10 ̊C•min. The heated apatite fibres were characterized by X-ray diffractometry (XRD), Fourier transform infrared spectrometry (FT-IR), scanning electron microscopy (SEM) and T EM. For TEM observation, bright and dark field imaging, combined with selected area electr on diffraction (SAED), was performed on a Jeol CX200 TEM operated at 200 kV. TEM samples were pr epared by dispersing the fibers in ethanol and collecting them onto lacey carbon, Cu mesh TEM grids. Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 915-918 doi:10.4028/www.scientific.net/KEM.254-256.915

Visualization of Carbon Nanoparticles Within Cells and Implications for Toxicity

Carbon nanostructures (CNS), such as C60, single-walled nanotubes (SWNTs) exhibit extraordinary properties and are one of the most commercially relevant class of NS. CNS have already found uses in high-performance sports equipment (nanotubes) and face cream (C60), whilst potential applications include optical and electronic materials and superconductors. Following the huge growth in these nanotechnology-related industries, significant concerns have arisen about their potential toxicity and impact on the environment. A lack in understanding of the interaction of such small structures with cellular material has resulted in concerns over their impact on human health. The potential toxicity of CNS and safety to human health requires an understanding of their interaction with cells and this in turn relies on the measurement of the pathways by which they enter the cell, their spatial distribution within and whether the CNS are transformed by the action of the cell; visualization of intracellular CNS is therefore imperative. However visualizing unlabelled CNS within cells is demanding because it is difficult to distinguish CNS from carbon-rich organelles given their similarity in composition and dimensions. In particular, the challenge lies in translating analytical imaging tools developed for inorganic systems to organic systems. This chapter describes how the state-of-the-art transmission electron microscopy (TEM) techniques, such as low-loss energy-filtered TEM (EFTEM) can be employed to differentiate between unlabelled C60, SWNTs and the cell. Further, we demonstrate how these techniques can be used to trace the uptake of CNS into the cell and to assess their localized effects on cell structure.