蛋白質的存在與作用為生命不可或缺的環節之一，不同功能的蛋白質，其形狀大小亦有差異。已知蛋白質的大小的一維範圍落在幾十奈米，然而光學顯微鏡受限於光波長繞射的限制，因此解析度只能到300 nm左右，蛋白質分子馬達(Protein-based molecular motor)、膜蛋白質離子通道(Membrane-embedded ion channels and transport proteins)和光合作用(Photosynthesis)等現象，無法有效地用光學顯微鏡來觀察，需用電子顯微鏡（Electron microscope）才有機會一窺蛋白質的原貌。受限於蛋白質組成元素為碳，氫，氧，硫，氮等較輕元素，在穿透式電子顯微鏡下，對比無法如金奈米粒子般清晰，因此需對樣品作切片等樣品處理，才能觀察到相關影像，我們使用氮化矽薄膜為基材之濕式腔體元件對具有已知特徵結構的蛋白質GroEL進行觀測，一般而言，氮化矽薄膜造成非彈性散射較碳膜（傳統穿透式電子顯微鏡樣品用）嚴重，我們針對此一問題進行改善，使用氫氟酸蝕刻，得到薄化後的wet-cell與碳膜有相似之解析能力，更能封住液體進行臨場(In situ)觀測的優點，更觀察到液態負染的影像，然而液態負染之影像並不清晰，因此改採更換背景基材的方式進行改善影像對比，並為未來wet-cell觀測視窗材料進行探索，目前使用矽薄膜作為基材，能觀察到鍵結在V-PPase上的金球對，未來希望利用軟體分析增強對比以及適當的背景材料觀測到未經人工處理的蛋白質影像。

The existence and functions are essential for life. Morphology and size are varied from different proteins. Many kinds of well-studied proteins are tens of nanometers in diameter, however, optical microscope, due to the limitation of wavelength of light, can offer only 300 nm in resolution and may not be suitable for observing such phenomenon as Protein-based molecular motor, Membrane-embedded ion channels and transport proteins and Photosynthesis and so forth. Thus, Electron microscope are required for observing proteins. Since proteins are composed of light elements such as C, H, O, S, N, which are not able to have intensive contrast as Gold nanoparticles do, frozen and staining treatments are needed. In this thesis, we use Si3N4 thin film as windows of our devices (Wet-cell) to observe a known protein with characteristic structure named GroEL. In fact, Si3N4 thin film will result in more inelastic scattered electrons than commercially used carbon film. To enhance the performance, we used HF to thin down Si3N4 membrane and acquired similar quality as acquired on carbon film. Furthermore, liquid samples can be sealed by Si3N4 membrane in order to In-situ observe the samples. Yet, we acquired some liquid state images which are not as clear as previous dry state images. Therefore, we plan to change background substrates to enhance images contrast and explore new materials for Wet-cell as well. Now we use Silicon as substrate and are able to visualize 1.8 nm Gold nanoparticle pairs labelled on the V-PPase. In the future, we hope to use software analysis to enhance contrast and find suitable materials to visualize protein images without artificial treatments.

摘要 i
Abstract ii
致謝 iii
目錄 v
表目錄 vii
圖目錄 viii
第1章 前言 1
1.1 蛋白質研究的挑戰 1
1.2 文獻回顧 2
1.2.1 近代液態穿透式電子顯微鏡的發展 2
1.2.2 生物樣品於液態電子顯微鏡觀測的發展 9
1.2.3 蛋白質於電子顯微鏡之相關研究 11
1.3 研究動機與目的 13
第2章 實驗原理與方法 14
2.1 藥品材料與儀器 14
2.1.1 Wet cell 製程材料與機台 14
2.2 樣品厚度對影像之影響 15
2.3 Wet-cell 濕式環境元件 17
2.3.1 Wet-cell 元件結構與設計概念 17
2.3.2 Wet-cell 元件製造 19
2.3.3 Wet-cell 元件改良 22
2.3.4 V-PPase蛋白質電鏡樣品製備 25
2.3.5 Wet-cell試片製備 25
第3章 結果與討論 27
3.1 乾燥蛋白質樣品於TEM下之觀測 28
3.1.1 矽基材之厚度影響 28
3.1.2 氮化矽薄膜厚度之影響 28
3.1.3 氮化矽薄膜薄化之原子力顯微鏡量測結果 30
表 3-1 蝕刻時間與理論厚度 30
3.1.4 氮化矽薄膜薄化之穿透式電子顯微鏡GroEL影像結果 38
3.2 濕式樣品在Wet-cell中之影像 43
3.2.1 Wet-cell液體封裝測試 43
表3-2 奈米金顆粒受液體影響之速度 45
3.2.2 GroEL 負染的液態影像 46
3.3 膜蛋白V-PPase之觀測 48
3.3.1 V-PPase 使用Wet-cell之負染結果 48
3.3.2 更換背景基材 50
3.4 影像解析度計算 54
第4章 結論與未來工作 56
4.1 結論 56
4.2 未來展望 57
參考文獻 58

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