本論文展示利用微機電技術，發展能強化電子顯微鏡影像對比之靜電式相位板，以拍攝未經染色之高分辨生物樣品影像。在傳統穿透式電子顯微鏡中，對生物試片進行拍攝，由於樣品通常為碳、氫、氧等與電子作用小的輕元素構成，需經過重金屬染色步驟，以增強影像上的吸收對比強度。但染劑與重金屬元素具有生物毒性，染色固定後細胞死亡且改變原有型態。根據Zernike的相位對比增強理論，應用Boersch提出的靜電式相位板概念，可於電子顯微鏡之後聚焦面改變穿透光與繞射光的相位差，即時增強影像的相位對比，無須對試片事先進行前處理，實現相位對比電子顯微鏡。本論文中，描述以微機電技術實現Boersch靜電式相位板的方法，提出改良型相位板晶片，具有自我檢測與局部加熱排除汙染的功能。相位板主體為三層金屬與兩層絕緣層構成的環形結構，以一根懸臂支撐橫跨在光圈上，對中間金屬電極進行偏壓，可調整通過環內的電子束相位，當相位差達到90度，將提升影像低頻區之相位對比。本計畫已初步獲得生物樣本之相位對比增強影像。在設計上透過加大光圈尺寸與縮小懸臂線寬，可減少相位板引發的背景吸收效應。絕緣層造成的電荷累積，會產生額外電場干擾成像，使用特殊的絕緣層內縮技術，可大為降低此效應對繞射圖形的扭曲。

This research purposes the development of phase contrast electron microscopy by a MEMS electrostatic phase plate, and the achievement to high resolution bio-sample imaging without staining. TEM provides high resolution images to in-organic materials due to high contrast of the in-organic materials to the supporting substrate materials when interacted with electron beams. In bio-image, most of the bio-specimen, containing mostly carbohydrate, needs staining to enhance the image contrast, due to the weak interactions between low Z atoms and incident electron beam. Zernike invented a phase plate to converse the phase information of transparent object to image intensity for getting high contrast images in optical microscopy. To obtain nm resolution for bio-molecule observation, Matsumoto proposed the employment of Boersch micro-lens (Electrostatic Zernike Phase Plate, EZPP) to shift the phase of a center electron beam to improve TEM phase images. The highest contrast occurs at the EZPP providing π/2 phase shift. We demonstrate an advanced version of the EZPP for protein visualization after the reduction of electron beam interaction. Lower the background absorption contrast by increasing the demension of the aperture and decreasing the width of the supporting cantilever. A special retraction design could avoid the electron charging to distort the diffraction pattern.

摘要 i
Abstract ii
誌謝 iii
目錄 iv
圖目錄 vi
Chapter 1. 相位板介紹 1
1.1. 何謂相位板 1
1.2. 為何需要發展相位板 3
Chapter 2. 相位板發展 12
2.1. 光學相位理論 16
2.2. 電子光學相位對比技術 19
2.3. 電子顯微鏡相位板 25
2.3.1. 磁場式相位板 25
2.3.2. 碳膜式相位板 28
2.3.3. 靜電式相位板 30
2.4. 新相位板設計特點 36
Chapter 3. 相位板設計與製作 39
3.1. 晶片設計 39
3.2. 製程步驟 44
3.2.1. 定義中間金屬層 47
3.2.2. 定義光圈開孔 48
3.2.3. 掏空基材 49
3.2.4. 定義下絕緣層 50
3.2.5. 覆蓋上絕緣層 50
3.2.6. 覆蓋外層金屬層 51
3.2.7. 金屬襯墊移除絕緣層 51
3.2.8. 挖穿中央空洞 52
3.3. 低光圈吸收對比相位板 53
Chapter 4. 相位板使用 54
4.1. 相位板適用範圍 54
4.2. 電性量測與施加偏壓 56
4.3. 相位對比顯微鏡系統 57
4.3.1. 穿透式電子顯微鏡系統 57
4.3.2. 預抽腔體、相位板Holder、三軸步進器 58
Chapter 5. 相位板製程討論 61
5.1. 降低吸收對比 – 增加光圈尺寸 61
5.2. 小線寬曝光良率 – 光罩與晶圓接觸 63
5.3. 金屬掀離良率 – 底切距離控制 66
5.4. 材料包覆良率 – 邊緣殘留翹起 69
5.5. 電性隔絕良率 – 氮化矽覆蓋 70
5.6. 聚焦離子束良率 – 幕簾效應 71
5.7. 減少電荷累積 – 側壁絕緣層露出 73
5.8. 減少電荷累積 – 環內絕緣層內縮 76
5.9. 減少電荷累積 – 氟碳化合物殘留 78
Chapter 6. 結論 79
參考文獻 80

[1]F. Zernike, Z. techn. Physik, vol. 16, pp. 454-457, 1935.
[2]R. Danev and K. Nagayama, Ultramicroscopy, vol. 88, pp. 243, 2001.
[3]H. Boersch, Z. Naturforsch, vol. 2a, pp. 615, 1947.
[4]Takao Matsumoto and Akira Tonomura, Ultramicroscopy, vol. 63, pp. 5-10, 1996.
[5]S.H. Huang et al., Journal of Electron Microscopy, vol. 55(6), pp. 273-280, 2006.
[6]E. Majorovits et al., Ultramicroscopy, vol. 107, pp. 213-226, 2007.
[7]Rossana Cambie et al., Ultramicroscopy, vol. 107, pp. 329-339, 2007.
[8]J. J. Bozzola and L. D. Russell, Electron Microscopy Principles and Techniques for Biologists, 2nd ed, Jones and Bartlett Publishers, Sudbury, MA., 1999.
[9]Douglas B. Murphy, Fundamentals of light microscopy and lectronic imaging, Wiley-Liss, (2001) 97
[10]Kuniaki Nagayama and Radostin Danev, Biophys Rev, vol. 1, pp. 37–42, 2009
[11]Scherzer O, J Appl Phys, vol. 20, pp. 20–29, 1949.
[12]L. Foucault, Ann. Obs. Imp. Paris, vol. 5, pp. 197, 1859.
[13]O. M. Rayleigh, Philos. Mag, vol. 33, pp. 161, 1917.
[14]A. Toepler: Ann. Phys. Chem, vol. 127, pp. 556, 1866.
[15]S. G. Lipson, H. Lipson and D. S. Tannhauser, Optical Physics, 3rd ed, Cambridge University Press, Cambridge, 1995.
[16]Kuniaki Nagayama, Journal of the Physical Society of Japan, vol. 73(10), pp. 2725, 2004.
[17]Danev R, Okawara H, Usuda N, Kametani K, Nagayama K, J Biol Phys, vol. 28, pp. 627–635, 2002.
[18]Danev R, Nagayama K, J Phys Soc Jpn, vol. 73, pp. 2718–2724, 2004.
[19]Schröder RR, Barton B, Rose H, Genner G, Microsc Microanal, vol. 13, pp. 136–137, 2007.
[20]Nagayama K, Eur Biophys J, vol. 37, pp. 345–358, 2008.
[21]Majorovits E, Barton B, Schultheiß K, Perez-Willard F, Gerthsen D, Schröder RR, Ultramicroscopy, vol. 107, pp. 213–226, 2007.
[22]Cambie R, Downing KH, Typke D, Glaeser RM, Jin J, Ultramicroscopy, vol. 107, pp. 329–339, 2007.
[23]Tomio Endo, Japan Patent, No. 607048(1985)
[24]Kuniaki Nagayama, Patent, No. 1950789 (2008)
[25]Kuniaki Nagayama et al., United States Patent, No. 6674078(2004)
[26]Kuniaki Nagayama., United States Patent Application Publication, No. 20080202918(2008)
[27]Takao Matsumoto et al., United States Patent, No. 5814815(1998)
[28]Takao Matsumoto et al., Ultramicroscopy 63, (1996) 5-10.
[29]Rasmus Schroder et al., Germany Patent, No. 10206703(2003)
[30]Gerd Benner., United States Patent , No. 6797956(2004)
[31]Kuniaki Nagayama and Radostin Danev, Phil. Trans. R. Soc. B, vol. 363, pp. 2153, 2008.
[32]ETH Zurich Electron Microscopy Site (http://www.microscopy.ethz.ch/)
[33]John C. H. Spence, High-Resolution Electron Microscopy, Oxford University Pres, 2008.
[34]Goodhew P J, Humphreys J and Beanland R, Electron Microscopy And Analysis, Taylor & Francis, pp. 76-121, 2001.
[35]Structure of Membrane Proteins and Protein Complexes by Cryo-EM (http://www.2dx.unibas.ch/)
[36]王婉稚, 黃森輝, 曾繁根, 陳福榮, 靜電式相位板在穿透式電子顯微鏡的應用, 2005.
[37]Olympus Microscopy Resource Center (http://www.olympusmicro.com/)
[38] Jessie Shiue, Chia-Seng Chang, Sen-Hui Huang et al, J Electron Microsc , pp. 1-9, 2009.