Transmission Electron Microscope Technique for Risk Assessment of Manufactured Nanomaterials

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1 Transmission Electron Microscope Technique for Risk Assessment of Manufactured Nanomaterials Kazuhiro Yamamoto and Miyabi Makino National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki , JAPAN 1. Introduction Manufactured applications of nano carbon materials such as fullerenes and carbon nanotubes are reported in many fields recently. The toxicity of these nano carbon materials for the human is not clear, therefore, the toxicity test and risk assessment are very important. Transmission electron microscope (TEM) is powerful technique to study the nano world, and is used both material and biological research. In the case of the observation for the biological cells, the cell specimens are usually stained with the heavy elements such as U and Pb to increase the contrast of image. However, it is difficult to observe the stained specimen of biological cells containing the nano-sized carbon materials, because the contrast from carbon is weak and below the background of the staining heavy elements. In this study, an energy-filtering TEM is applied to the in-vivo test of fullerene nano particles. The lung tissue specimen after the intratracheal instillation of fullerenes solution in the rat lung is observed. 2. Materials and Methods 2.1 Energy-filtering TEM The interaction between the electron and the specimen is shown in Fig. 1. Most of the primary incident electrons are through the specimen or elastically scattered, which are shown with black lines. A part of electrons, which are shown with gray lines, are inelastically scattered and lose the energies due to the various inelastic process. These energy loss electrons cause the decrease of the contrast and the unclear image. Therefore the high contrast observation is achieved by making images using only the energy zero-loss electrons. This technique is called the zero-loss imaging [Ref.1]. Zero-loss filtering can increase the scattering and phase contrast of TEM image. The zero-loss imaging was performed by EM922 (Carl Zeiss NTS, Germany), which was equipped OMEGA energy filter. The energy window-width of the filter was 20 ev, and the acceleration energy was 200kV.

2 2.2 Experimental The in-vivo test of fullerenes was performed using nine-week-old male Wister rats. The test solution was the fullerene nano particles in the water with 0.1g/l tween 80 dispersions. The fullerenes solutions of 0.1mg / 0.4ml or 0.2mg / 0.4ml content were intratracheally instilled into the rat lung. The TEM images of the fullerene solution are shown in Fig. 2 (a) and (b). The diameter of fullerenes is 30 nm, and the fullerene is crystalline. The lung tissues at one week, one month, and three months post-instillation were observed by TEM. The preparation method of TEM specimen was as follows. The lung tissue were fixed using glutaraldehyde and osmium

3 tetroxide solution, and then dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were cut on a diamond knife with microtomy. The staining was performed by using 2% uranyl acetate solution and 0.5% lead citrate solution at room temperature. The staining time for each staining solution was examined. 3. Results Low magnification image of the rat lung tissue at 1 week after the instillation exposure is shown in Fig. 3. The alveolar cells and the alveolar macrophages are observed. The zero-loss image of the alveolar macrophages at 1 week post-instillation is shown in Fig. 4 (a). The some particles with the black contrast are observed at the cytoplasm. These particles are not observed in the nucleus or the mitochondria. The high-resolution image of these particles is shown in Fig. 4 (b). The lattice structure is observed and it is clarified that this particle is crystalline. The selected area electron diffraction pattern of these particles and that of fullerene nano-particles in test solutions are shown in Fig. 5 (a) and (b), respectively. Both diffraction patterns are in good agreement; therefore the black particles observed in the alveolar macrophages are fullerenes.

4 Fullerenes particles keep the fcc crystal structure in the cells. As the diameter of fullerenes in Fig. 4 (b) is 30 nm, the fullerenes keep the particle size in the macrophages. The most of fullerenes particles are observed in the alveolar macrophages, however, some fullerenes are observed in the alveolar cells. Fullerenes still remains in the alveolar macrophages and the alveolar cells at 1 month or 3 months post-instillation. 4. Summary Zero-loss imaging by using the energy-filtering TEM was applied to the in-vivo test of

5 fullerene nano particles. The fullerenes solution is intratracheally instilled into the rat lung. The lung tissue specimen at 1 week, 1 month, and three months post-instillation were examined. The fullerene particles are observed in the cytoplasm of the alveolar macrophages at 1 week post-instillation. The fullerenes are crystalline and keep the diameter of 30 nm. Some fullerenes are observed in the alveolar cells. Fullerenes still remains in the alveolar macrophages and the alveolar cells at 1 month or 3 months post-instillation. It is necessary to investigate the trace of these fullerenes in our future work. 5. References Reimer, L.: Transmission Electron Microscopy 3 rd Berlin, 1993) Edition; pp (Springer-Verlag: