臺灣博碩士論文加值系統

本研究利用微機電技術開發出ㄧ種新穎且可拋棄式的微元件（命名為K-kit），做為一種裝載試片的裝置，K-kit能使活體生物在液態環境下可直接於臨場穿透式電子顯微鏡進行觀察，其中K-kit包含電子束可穿透的二氧化矽奈米薄膜，並且可使用於不需改裝之穿透式電子顯微鏡。本研究經模擬指出，當入射電子束電流密度為30 pA/cm2，對於磷鎢酸負染色且密封於K-kit內的活體的細菌，其解析度大約可達2.2奈米，實驗上透過K-kit，成功的利用穿透式電子顯微鏡，觀察到活體的大腸桿菌、寬8到18奈米成束的活體克雷白氏肺炎桿菌第三型線毛，以及克雷白氏肺炎桿菌還原碲金屬的過程。
在拍攝穿透式電子顯微鏡影像之前，實驗發現克雷白氏肺炎桿菌可在密封於K-kit 12小時後，具有超過八成的存活率。除此之外，我們也試驗密封在K-kit的克雷白氏肺炎桿菌（革蘭氏陰性菌）和啤酒酵母菌經過電子束照射後的存活能力，研究發現在穿透式電子顯微鏡的電子束連續照射下，多數的克雷白氏肺炎桿菌可存活約14秒，而啤酒酵母菌則可存活約42秒。藉由電子能量損失能譜儀之量測數據，本研究計算出電子損失於克雷白氏肺炎桿菌和啤酒酵母菌的能量分別為329.1 eV nm-3 and 405.3 eV nm-3，經過比較其表面構造後，發現啤酒酵母菌具有較緊密且共價鍵結的細胞壁，克雷白氏肺炎桿菌的外膜則是為較鬆散的夾膜多醣體所構成，可能是導致克雷白氏肺炎桿菌顯示出比啤酒酵母菌更容易受電子照射所影響的原因。
本研究更進一步利用K-kit在穿透式電子顯微鏡下，觀察克雷白氏肺炎桿菌還原碲金屬的生物反應長達12.5小時，比較在有氧及厭氧的環境下有明顯不同碲金屬還原的樣貌。經過以上的實驗結果證實，本研究開發的K-kit確實可以應用於臨場穿透式電子顯微鏡，於濕式環境下，進行活體生物觀察和即時的生物反應觀測。

A novel and disposable microchip (named as K-kit) with electron-transparent SiO2 nano-membranes was developed using microelectromechanical system techniques and used as a specimen kit for in situ imaging of living organisms in an aqueous condition by transmission electron microscopy (TEM) without equipment modification. The resolving power of living bacterial cells negatively stained with phosphotungstic acid was theoretically calculated to be 2.2 nm in K-kit at incident current density of 30 pA/cm2. Experimentally, this K-kit which can enclose aqueous specimens enabled the successful TEM observation of living Escherichia coli cells, the 8-18 nm type 3 fimbriae of living Klebsiella pneumoniae, and the tellurite reduction process in K. pneumoniae in-situ.
The survival ratio of K. pneumoniae sealed in the K-kit for 12 h exceeded 80% before TEM imaging. Besides, the viability of bacterial cells sealed in the K-kits during TEM electron irradiation was examined. The K. pneumoniae (gram-negative bacteria) and Saccharomyces cerevisiae (yeast cells) can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Utilizing the measurement by electron energy-loss spectroscopy, the total electron energy of 329.1 eV nm-3 and 405.3 eV nm-3 dissipated in K. pneumoniae　and S. cerevisiae were calculated, respectively. By the comparison of their surface structures, S. cerevisiae cells exhibit more dense-packed and covalently linked cell walls than the loosely attached capsular polysaccharides on the outer membrane of K. pneumoniae. It is possible that K. pneumoniae is more susceptible to electron beam irradiation compared to S. cerevisiae.
Furthermore, the bio-reaction, tellurite reduction in K. pneumonia, was in situ monitored by TEM for 12.5 h through the use of the K-kit. The different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be also clearly revealed. These results demonstrate that the K-kit developed in this study can be useful for observing living organisms and in situ monitoring bio-reaction.

摘要……………………. i
Abstract……. iii
誌謝………… v
Contents……. viii
List of Figures xi
List of Tables xv
Chapter 1 Introduction 1
Chapter 2 Literature Review 4
2.1 Biological Transmission Electron Microscopy 4
2.1.1Conventional transmission electron microscopy……………...4
2.1.2Cryo-transmission electron microscopy (Cryo-TEM)………...5
2.2 Modification of Electron Microscopes for Observation of Wet Samples. 6
2.2.1 Environmental transmission electron microscopy (ETEM) 6
2.2.2 Scanning electron microscopy for wet biological specimens .9
2.3 Specimen Damage by Electron Beam Irradiation 11
2.3.1 Electron-specimen interaction……………………………….11
2.3.2 Damage processes in materials……………………………...12
Chapter 3 Theory of TEM Resolution and Calculation of Electron Energy Loss 13
3.1 Signal to Noise Ratio and Rose’s Criteria for TEM Resolution……… 13
3.2 Calculation of Electron Energy Loss in the Specimen 15
3.3 Log-Ratio Formula for Calculation of Specimen Thickness 15
Chapter 4 Experimental 19
4.1 Process Flow………………………………………………………19
4.2 K-kit Microchip Fabrication 21
4.3 Cell Cultures 24
4.3.1 Bacteria and Yeast Cell Cultures 24
4.3.2 Human hepatocyte (HepG2) culture 25
4.4 Cell Stain 25
4.4.1 Fluorescent stain of cells for fluorescent microscopy 25
4.4.2 Negative stain of bacterial cells for TEM imaging 26
4.5 Tellurium Reduction by Klebsiella pneumoniae 27
4.5.1 Sample preparation of tellurite reduction in K. pneumoniae for TEM imaging 27
4.5.2 Sealing the K. pneumoniae in K-kit for Long-term and in situ imaging of tellurite reduction by TEM M 28
Chapter 5 Experimental Instruments and Operation 29
5.1 Transmission Electron Microscope (TEM) 29
5.1.1 Minimum exposure operation………………..……………...29
5.1.2 Elecron energy loss spectroscope (EELS) 31
Chapter 6 Result and Disscussion 32
6.1 TEM Specimen Kit (K-kit) Design and Fabrication 32
6.2 Survival Ratio of the Cells Sealed in K-kit 37
6.3 Viability of Bacterial Cells after TEM Imaging 42
6.4 TEM Resolution of Living Cells under Aqueous Conditions 44
6.5 Viability of Bacterial Cells Exposed to the Electron Beam for Various Durations 48
6.6 Electron Energy Dissipation in Living Cells Measured using In-situ EELS 53
6.7 The Susceptibility of Living Cells to Electron Beam Irradiation 63
6.8 In situ TEM Observation of Tellurite Reduction by K. pneumoniae …………………………………………………………………..65
6.8.1 Characterization of tellurite-nanopartical reduction in K. pneumoniae using TEM 65
6.8.2 In situ imaging of tellurite reduction in K. pneumoniae by TEM 67
Chapter 7 Conclusion 71
Chapter 8 Future Prospects 74
Reference ……………………………………………………………….76
List of Publications 91

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