磁小體(Magnetosomes)在磁性細菌(magnetotactic bacteria)內之成長於生物科學及醫學仿生材料上有重要意義，如基因治療與癌症熱治療等。但迄今為止其成長機制仍未有定論。傳統上多以冷凍切片方式觀測磁性細菌與磁小體，因此難於觀測合成過程。本研究以臨場觀測磁小體成長過程為目標，據此設計一套實驗方式，其中包含(1)微機電製程設計應用於流體環境元件(Flow cell)開發與(2)穿透式電子顯微鏡(TEM)空錐暗場(Hollow Cone Dark Field)應用於液體環境拍攝。
於Flow cell因不同樣品之厚度需求，利用(1)金屬蒸鍍或(2)光阻塗佈設計墊高層(Spacer)控制液體厚度與空間，配合流體系統樣品台(liquid holder)，可提供養份與移除代謝物，並臨場觀察樣品變化。
為解決TEM拍攝生物樣品與生俱來的弱對比問題(來自樣品內的低原子序碳氫氧原素)因此利用TEM的兩組中間透鏡系統，將入射電子偏移離開中央軸，產生偏光空錐暗場(Tilt illumination Hollow Cone Dark Field)使影像對比增加。
現階段成功於流體系統中，觀察到磁小體受電子與水影響而溶解的過程，並利用空錐暗場於乾式環境拍攝磁性細菌鞭毛，鞭毛部分對比與明場相比大幅提升十一倍，於流體環境細菌影像對比亦提升四倍。

Growth of magnetosomes in magnetotactic bacteria is important in the biological sciences and application of medical biomimetic materials, such as gene therapy and cancer heat treatment. However, the growth mechanism is still inconclusive so far. Traditionally, magnetotactic bacteria and magnetosomes have previously been studied by cryo thin sections method, which is difficult to directly observe the synthesis process. In this study, we aim to observe the process of magnetosome synthesis, and according to this purpose to design a series of experiments, including (1) Application of MEMS process design on Flow cell development and (2) Application of Hollow cone dark field (HCDF) on photographing objects in liquid environment in TEM.
In Flow cell, we use (1) metal deposition, or (2) photoresist coating method to design different thickness spacers to control the thickness of the liquid and space. With combination of Flow cell and liquid holder, we can provide an open system for bacteria.
In order to solve the weak contrast of biological samples (arising from low atomic number elements of sample) , we use tilt illumination hollow cone dark field by adjusting two lens system to shift the incident electron from the central axis to increase the image contrast.

目錄
摘要………………………………………………………………………………………i
目錄…………………………………………………………...…………………………ii
第一章 前言…………………………………………………………………………….1
1.1 生物樣品研究之挑戰………………………………………………………1
1.2 文獻回顧……………………………………………………………………3
1.2.1 近代液態電子顯微鏡發展 ………………………………………..3
1.2.2 磁性細菌……………………………………………………………9
1.2.3 磁小體……………………………………………………………..11
1.2.4 磁性細菌之應用………………………………………………….12
1.2.5 電子顯微鏡觀察磁性細菌………………………………………..12
1.2.6 環形暗場穿透式電子顯微鏡………………………………….…13
1.2.7 空錐暗場穿透式電子顯微鏡……………………………………18
第二章 實驗方法與原理……………………………………………………………...20
2.1 藥品材料與儀器……………………...……………………………………20
2.1.1 細菌培養之材料與觀測機台……………………………………..20
2.1.2 濕式環境元件製程材料與機台…………………………………..23
2.2 磁性細菌之培養與樣品製備……………………………………………...25
2.2.1 培養基配製方法…………………………………………………..25
2.2.2 冷凍保存之配方與配製…………………………………………..27
2.2.3 磁性細菌活化與培養……………………………………………..28
2.2.4 磁性細菌樣品製備……………………………………… .……..29
2.3 Wet cell濕式環境元件………………………………………….………30
2.3.1 wet cell設計概念……………………………………………….30
2.3.2 SAW cell元件製程…………………………………………...…33
2.3.3 wet cell 元件改良………………………………………………38
2.4 濕式TEM樣品製備……………………………………………………45
2.4.1 利用SAW cell 封裝樣品流程………………………………….46
2.4.2 利用Out-frame對貼封裝樣品流程……………………….…….47
第三章 結果與討論…………………………………………………………………...48
3.1 流體環境元件設計………………………………………………………….48
3.1.1 流體環境元件設計……………………………………………………..48
3.1.2 金屬墊高層……………………………………………………………..50
3.1.3 光阻電高層……………………………………………………………..52
3.2 磁菌樣品製備……………………………………………………………….55
3.2.1 磁性細菌汙染問題……………………………………………………..55
3.2.2 磁性細菌與培養液……………………………………………………..57
3.3 利用電子顯微鏡觀察………………………………………………………59
3.3.1 利用掃描式電子顯微鏡觀察磁性細菌……………………………….59
3.3.2 利用穿透式電子顯微鏡觀察磁性細菌……………………………….60
3.3.3 細菌與晶片之相容性………………………………………………….63
3.3.4 封閉式問題討論……………………………………………………….64
3.3.5 流體系統觀測磁性細菌…………………………………………………..67
3.3.6 磁小體受電子與液體影響溶解…………………………………………..70
第四張 結論…………………………………………………………………………...74
參考文獻……………………………………………………………………………….75

[1] M. J. Williamson, R. M. Tromp, P. M. Vereecken, R. Hull, and F. M. Ross, “Dynamic microscopy of nanoscale cluster growth at the solid-liquid interface.,” Nat. Mater., vol. 2, no. 8, pp. 532–536, 2003.
[2] a. Radisic, F. M. Ross, and P. C. Searson, “In situ study of the growth kinetics of individual island electrodeposition of copper,” J. Phys. Chem. B, vol. 110, no. 15, pp. 7862–7868, 2006.
[3] U. Dahmen and A. P. Alivisatos, “Reports 13. 14.,” Science (80-. )., vol. 324, pp. 1309–1312, 2006.
[4] N. de Jonge, N. Poirier-Demers, H. Demers, D. B. Peckys, and D. Drouin, “Nanometer-resolution electron microscopy through micrometers-thick water layers.,” Ultramicroscopy, vol. 110, no. 9, pp. 1114–9, Aug. 2010.
[5] X. Chen and J. Wen, “In situ wet-cell TEM observation of gold nanoparticle motion in an aqueous solution,” Nanoscale Res. Lett., vol. 7, no. 1, p. 598, 2012.
[6] J. M. Yuk, J. Park, P. Ercius, K. Kim, D. J. Hellebusch, M. F. Crommie, J. Y. Lee, a Zettl, and a P. Alivisatos, “High-Resolution EM of Colloidal Nanocrystal Growth Using Graphene Liquid Cells,” Science, vol. 336, no. 6077. Aaas, pp. 61–64, 2012.
[7] B. Richard p, “MAGN’ETOTACTIC BACTERIA,” Science (80-. )., vol. 190, pp. 217–238, 1982.
[8] D. a Bazylinski and R. B. Frankel, “Magnetosome formation in prokaryotes.,” Nat. Rev. Microbiol., vol. 2, no. 3, pp. 217–230, 2004.
[9] Y. Liu, M. Gao, S. Dai, K. Peng, and R. Jia, “Characterization of magnetotactic bacteria and their magnetosomes isolated from Tieshan iron ore in Hubei Province of China,” Mater. Sci. Eng. C, vol. 26, no. 4, pp. 597–601, 2006.
[10] K. L. Thomas-Keprta, S. J. Clemett, D. a Bazylinski, J. L. Kirschvink, D. S. McKay, S. J. Wentworth, H. Vali, E. K. Gibson, M. F. McKay, and C. S. Romanek, “Truncated hexa-octahedral magnetite crystals in ALH84001: presumptive biosignatures.,” Proc. Natl. Acad. Sci. U. S. A., vol. 98, no. 5, pp. 2164–2169, 2001.
[11] a Gorby, T. J. Beveridge, and R. P. Blakemore, “Characterisation of the bacterial magnetosome,” J. Bacteriol., vol. 170, no. 2, pp. 834–841, 1988.
[12] A. Komeili, “Molecular mechanisms of compartmentalization and biomineralization in magnetotactic bacteria,” FEMS Microbiol. Rev., vol. 36, no. 1, pp. 232–255, 2012.
[13] J. Li and Y. Pan, “AMB-1: Implications for Biologically Controlled Mineralization,” Geomicrobiol. J., vol. 29, no. 4, pp. 362–373, 2012.
[14] D. Faivre, N. Menguy, M. Pósfai, and D. Schüler, “Environmental parameters affect the physical properties of fast-growing magnetosomes,” Am. Mineral., vol. 93, no. 2–3, pp. 463–469, 2008.
[15] “Bazylinski.MicrosResTech.1994.pdf.” .
[16] M. Bacteria and M. Program, “Magnetotactic Bacteria and,” no. September, pp. 4875–4898, 2010.
[17] D. Schüler and R. B. Frankel, “Bacterial magnetosomes: Microbiology, biomineralization and biotechnological applications,” Appl. Microbiol. Biotechnol., vol. 52, no. 4, pp. 464–473, 1999.
[18] F. D. M. A.S.Bahaj, P.A.B.James, “Wastewater Treatment By Bio-Magnetic Separation:A Comparison of Iron Oxide And Iron Sulphide Biomass Recovery.” Wat.Sci.Tech., pp. 311–317, 1998.
[19] a. S. Bahaj, P. a. B. James, and F. D. Moeschler, “Continuous radionuclide recovery from wastewater using magnetotatic bacteria,” vol. 184, pp. 241–244, 1998.
[20] A. Arakaki, H. Takeyama, T. Tanaka, and T. Matsunaga, “Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus RS-1, using magnetic separation.,” Appl. Biochem. Biotechnol., vol. 98–100, pp. 833–840, 2002.
[21] F. Cai, J. Li, J. Sun, and Y. Ji, “Biosynthesis of gold nanoparticles by biosorption using Magnetospirillum gryphiswaldense MSR-1,” Chem. Eng. J., vol. 175, no. 1, pp. 70–75, 2011.
[22] Z. Lu and S. Martel, “Preliminary investigation of bio-carriers using magnetotactic bacteria,” Annu. Int. Conf. IEEE Eng. Med. Biol. - Proc., pp. 3415–3418, 2006.
[23] Q. a Pankhurst, J. Connolly, S. K. Jones, and J. Dobson, “Applications of magnetic nanoparticles in biomedicine,” J. Phys. D …, vol. 167, no. 13, pp. R167–R181, 2003.
[24] E. Alphandéry, F. Guyot, and I. Chebbi, “Preparation of chains of magnetosomes, isolated from Magnetospirillum magneticum strain AMB-1 magnetotactic bacteria, yielding efficient treatment of tumors using magnetic hyperthermia,” Int. J. Pharm., vol. 434, no. 1–2, pp. 444–452, 2012.
[25] E. Alphandéry, S. Faure, O. Seksek, F. Guyot, and I. Chebbi, “Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy,” ACS Nano, vol. 5, no. 8, pp. 6279–6296, 2011.
[26] T. J. Woehl, S. Kashyap, E. Firlar, T. Perez-Gonzalez, D. Faivre, D. Trubitsyn, D. a. Bazylinski, and T. Prozorov, “Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging,” Sci. Rep., vol. 4, p. 6854, 2014.
[27] R. Aso, H. Kurata, T. Namikoshi, T. Hashimoto, S. W. Kuo, F. C. Chang, H. Hasegawa, M. Tsujimoto, M. Takano, and S. Isoda, “Quantitative imaging of T g in block copolymers by low-angle annular dark-field scanning transmission electron microscopy,” Macromolecules, vol. 46, no. 21, pp. 8589–8595, 2013.
[28] O. Scherzer, “The theoretical resolution limit of the electron microscope,” J. Appl. Phys., vol. 20, no. 1, pp. 20–29, 1949.
[29] G. Dupouy, “Contrast improvement in electron microscopic images of amorphous objects.,” J. Electron Microsc. (Tokyo)., vol. 16, no. 1, pp. 5–16, 1967.
[30] S. Bals, G. Van Tendeloo, and C. Kisielowski, “A new approach for electron tomography: Annular dark-field transmission electron microscopy,” Adv. Mater., vol. 18, no. 7, pp. 892–895, 2006.
[31] F. Leroux, E. Bladt, J.-P. Timmermans, G. Van Tendeloo, and S. Bals, “Annular dark-field transmission electron microscopy for low contrast materials.,” Microsc. Microanal., vol. 19, no. 3, pp. 629–34, 2013.
[32] “._1981-Hollow cone illumination—I. Dark-field electron microscopy of biological specimens.pdf.” .
[33] T. Geipel and W. Mader, “Practical aspects of hollow-cone imaging,” Ultramicroscopy, vol. 63, no. 1, pp. 65–74, 1996.
[34] W. Krakow and L. a Howland, “A method for producing hollow cone illumination electronically in the conventional transmission microscope.,” Ultramicroscopy, vol. 2, no. 1, pp. 53–67, 1976.
[35] B. Yao, T. Sun, A. Warren, H. Heinrich, K. Barmak, and K. R. Coffey, “High contrast hollow-cone dark field transmission electron microscopy for nanocrystalline grain size quantification,” Micron, vol. 41, no. 3, pp. 177–182, 2010.
[36] J. M. Grogan, L. Rotkina, and H. H. Bau, “In situ liquid-cell electron microscopy of colloid aggregation and growth dynamics,” Phys. Rev. E, vol. 83, no. 6, p. 061405, Jun. 2011.
[37] N. M. Schneider, M. M. Norton, B. J. Mendel, J. M. Grogan, F. M. Ross, and H. H. Bau, “Electron−Water Interactions and Implications for Liquid Cell Electron Microscopy,” 2014.