電子顯微鏡已經是材料科學領域中的一項不可或缺的研究工具，近年來隨著科學不斷的進步，材料以及生物領域的研究倍受重視，由於生物活體細胞隨時可能發生變異或者死亡，因此可以快速尋標定位的光學顯微鏡將可做為電子顯微鏡的輔助工具，本論文設計一個光學顯微鏡與電子顯微鏡結合的系統，我們稱此系統為Correlative Light and Electron Microscope(CLEM)。最主要之目的在於以低倍率光學顯微鏡先尋找樣品目標並定位後續細部量測點，再切換以高倍率電子顯微鏡做定點細部觀察。
CLEM已漸漸成為近年來顯微檢測領域的一項重要利器，市面上各大電子顯微鏡公司陸續推出商品化機台，而各廠家的設計架構各自有其使用上的優缺點。本論文研究開發出一套創新的CLEM系統，其特色在於光學顯微鏡之光路與電子顯微鏡之電子束於入射樣品時共用同一路徑，同時皆是由樣品上方觀察樣品，有別於大部分商用機台是將光學顯微鏡光路與電子顯微鏡之電子束路徑分別設計在樣品的上、下側。目前我們已經成功地將光學顯微鏡模組與掃描式電子顯微鏡之真空腔體整合並且實際進行兩種模式的觀察，能夠從光學顯微鏡觀察模式即時切換到電子顯微鏡模式做樣品的細部觀察。如此一來，本論文設計之CLEM系統所觀察到的樣品影像將除了具有從10 倍到10,000倍的功能外，也同時具備了不同倍率影像之間的高重複性以及關聯性。

We have developed a new CLEM (Correlative Light Electron Microscope) with a merged system combining a light microscope module and a commercial desktop electron microscope (TEMIC-EM100). A 10X objective lens with long working distance is positioned in between the BSED (Backscatter Electron Detector) and the sample stage of a scanning electron microscope (SEM). A 45 mirror mounted on a home-built objective nose piece guides the light from the objective lens to the sample and vice versa. The system is designed in such a way that all components of the light microscope except the objective lens and the reflection mirror are positioned outside the vacuum. With the new system development, the SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample by simply moving and repositioning the light microscope module, thus leading to correlative imaging.
We demonstrate the use of our light microscope module as a kind of “preview option” for SEM. The light microscope gives visibility to the copper mesh (grid size : 5 μm*5 μm), individual grid can be clearly identified in the image. Based on the image, a region can be selected for SEM inspection at high magnification.
Our future work is to extend a fluorescence analysis function in the module to study living cells. We have developed a fluorescence microscope module on the original low magnification “preview” module. A 350 W Xenon lamp light source with collimator lens is used to illuminate polystyrene particle sample(Fluoro-Max R400 ; particle size 400 nm), and an emission of orange 612 nm fluorescence image is taken by the 100X objective lens.

摘要 I
Abstract II
致謝 III
總目錄 VI
圖目錄 VIII
表目錄 XIII
第一章 前言 1
1.1顯微鏡發展史 1
1.2研究動機與目的 3
第二章 文獻回顧 5
2.1 早期光學顯微鏡與電子顯微鏡整合 5
2.2近期螢光顯微鏡與SEM整合與應用 10
第三章 實驗設計與方法 16
3.1光學顯微鏡模組之光路設計 16
3.1.1光學顯微鏡模組之光路設計 16
3.1.2光學顯微鏡與電子顯微鏡腔體整合設計 18
3.1.3螢光顯微鏡模組之光路設計 20
3.2材料與儀器 22
3.2.1光學顯微鏡套件 22
3.2.2校正治具 28
3.2.3整合系統 30
3.2.4 螢光顯微鏡套件 32
3.3光學顯微鏡模組校正與規格確立 39
3.3.1光學顯微鏡之光路校正 39
3.3.2光學顯微鏡模組光路校正結果 44
3.3.3 Field of View規格確立 48
第四章 結果與討論 49
4.1 CLEM成品 49
4.1.1 CLEM外觀 49
4.1.2 CLEM系統真空測試 52
4.1.3 光學影像品質最佳化 56
4.1.4 CLEM影像 59
4.2 fluorescence SEM (FL-SEM) 63
4.2.1 光學物鏡取像 63
4.2.2 螢光顯微鏡模組取像 69
4.2.3 FL-SEM整合 73
4.3 結論與未來發展方向 76
第五章 參考文獻 77

[1] J. Caplan, M. Niethammer, R. M. Taylor II, and K. J. Czymmek, "The power of correlative microscopy: multi-modal, multi-scale, multi-dimensional," Current opinion in structural biology, vol. 21, pp. 686-693, 2011.
[2] A. V. Agronskaia, J. A. Valentijn, L. F. van Driel, C. T. Schneijdenberg, B. M. Humbel, P. M. van Bergen en Henegouwen, et al., "Integrated fluorescence and transmission electron microscopy," Journal of structural biology, vol. 164, pp. 183-189, 2008.
[3] B. N. Giepmans, "Bridging fluorescence microscopy and electron microscopy," Histochemistry and cell biology, vol. 130, pp. 211-217, 2008.
[4] S. S. K. Ohsawa, "Electron microscope with optical microscope," United States Patent, p. 4266128, 1979.
[5] A. Kazumichi Ogura, "Specimen observation apparatus including an optical microscope and a scanning electron microscope," United States Patent, p. 4349242, 1981.
[6] J. S. Faber, "Charged particle instrument equipped with optical microscope," United States Patent, pp. 7,675,034 B2, 2010.
[7] A. C. Zonnevylle, R. F. Van Tol, N. Liv, A. C. Narvaez, A. P. Effting, P. Kruit, et al., "Integration of a high-NA light microscope in a scanning electron microscope," J Microsc, vol. 252, pp. 58-70, Oct 2013.
[8] C. Wouters and H. Koerten, "Combined light microscope and scanning electron microscope, a new instrument for cell biology," Cell biology international reports, vol. 6, pp. 955-959, 1982.
[9] T. Takagi, "Scanning electron microscope with an optical microscope," United States Patent, pp. 4,537,477, 1985.
[10] J. W. Lichtman and J.-A. Conchello, "Fluorescence microscopy," Nature methods, vol. 2, pp. 910-919, 2005.
[11] R. Yuste, "Fluorescence microscopy today," Nature methods, vol. 2, pp. 902-904, 2005.
[12] W. Kukulski, M. Schorb, S. Welsch, A. Picco, M. Kaksonen, and J. A. Briggs, "Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision," The Journal of cell biology, vol. 192, pp. 111-119, 2011.
[13] T. Kanemaru, K. Hirata, S. Takasu, S. Isobe, K. Mizuki, S. Mataka, et al., "A fluorescence scanning electron microscope," Ultramicroscopy, vol. 109, pp. 344-9, Mar 2009.
[14] S. Watanabe, A. Punge, G. Hollopeter, K. I. Willig, R. J. Hobson, M. W. Davis, et al., "Protein localization in electron micrographs using fluorescence nanoscopy," Nature methods, vol. 8, pp. 80-84, 2011.
[15] H. Nishiyama, M. Suga, T. Ogura, Y. Maruyama, M. Koizumi, K. Mio, et al., "Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film," J Struct Biol, vol. 172, pp. 191-202, Nov 2010.
[16] Y. Maruyama, T. Ebihara, H. Nishiyama, M. Suga, and C. Sato, "Immuno EM–OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM)," Journal of structural biology, vol. 180, pp. 259-270, 2012.
[17] B. G. Kopek, G. Shtengel, C. S. Xu, D. A. Clayton, and H. F. Hess, "Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes," Proceedings of the National Academy of Sciences, vol. 109, pp. 6136-6141, 2012.
[18] 蔡濂聲, "Correlative顯微鏡之研發與應用," 工業技術研究院 量測技術發展中心 量測資訊雙月刊157期, p. 29, 2014.