穿透式電子顯微鏡之空錐照射暗場像於生物樣品之應用

Hollow Cone Electron Imaging for Biological Materials

蔡俊穎(Tsai, Chun Ying)

指導教授 ： 陳福榮

國立清華大學/原子科學院/工程與系統科學系/博士(2016年)

摘要

本論文主要探討以收集生物樣品之熱漫散射(Thermal Diffuse Scattering)電子的”空錐照射暗場像 (Hollow Cone Dark Field, HCDF)”來增強生物樣品在電子顯微鏡中的對比，並針對三種生物樣品製備方式進行實驗。在本論文中，就生物樣品製備可以分成三大部分：（一）負染生物樣品，（二）低溫生物樣品，（三）臨場濕式生物樣品。在第一部份我們先以醋酸鈾醯負染GroEL蛋白質做為樣品，針對負染GroEL蛋白質找出空錐照射暗場像之最佳化拍攝條件並證明空錐照射暗場像能夠提升生物樣品對比，最後藉由單分子三維重構技術重建出GroEL 13.57Å三維結構。在第二部分中，我們開發出一套應用於低溫電子顯微鏡之空錐照射暗場像拍攝流程，成功取得微脂體約2奈米的磷脂雙層膜。接著我們拍攝幾種低溫生物樣品，包含：KLH (Keyhole Limpet Hemocyanin)蛋白質、腺病毒與魚類神經壞死病毒，並驗證HCDF能夠提升約四倍對比。同時，對NNV病毒影像進行單分子三維重構，發現三維重構結果無法收斂並探討其原因。第三個部分是以自行開發的臨場濕式樣品桿與臨場濕式晶片觀察磁性細菌(Magnetospirillum magnetotacticum, MS-1)，以磁性細菌中的磁小體來定位細菌位置。從影像上得知HCDF對比約為明場像之五倍，且可觀察到較清楚之細菌外型。

關鍵字

空錐照射暗場像 ； 穿透式電子顯微鏡 ； 單分子重構 ； 臨場濕式電子顯微鏡 ； 低溫電子顯微鏡

並列摘要

In this dissertation, we present a new route to achieve the same goals by hollow cone dark field (HCDF) imaging using thermal diffuse scattered (TDS) electrons giving about a 4 times contrast increase as compared to bright field imaging It can be divided into three topics: (1) negative staining, (2) Cryo-electron microscopy, and (3) in situ liquid cell TEM. In the first part, we demonstrated the optimum condition of HCDF and contrast enhancement by negative stained GroEL. After that, we demonstrated the 3D reconstruction of a negative stained GroEL particle can yield about 13.5Å resolution but using a strongly reduced number of images. In the second part, we established the process flow of HCDF technique for Cryo-EM field and demonstrated the concept by liposome. Later, we demonstrated the first result utilized HCDF technique in various biological sample including Keyhole Limpet Hemocyanin (KLH) protein, Adenovirus and Nervous Necrosis Virus (NNV) and the 3D reconstruction model of NNV. In the last part, we demonstrated the HCDF in in situ liquid TEM field and observed the Magnetospirillum magnetotacticum, (MS-1). We proved the HCDF technique provided contrast enhancement and more detail than tradition bright field mode.

並列關鍵字

Hollow Cone Dark Field ； Transmission Electron Microscopy ； Single Particle Reconstruction ； in situ Liquid EM ； Cryo EM

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