開發穿透式電子顯微鏡流體系統: 臨場觀察磁性細菌之磁小體成長

Development of Flow Environment TEM for In-Situ Investigating the Mechanism of Magnetesome Growth in Magnetic Bacteria

翁宇弘(Weng,Yu Hung)

指導教授 ： 陳福榮 ； 曾繁根

國立清華大學/原子科學院/工程與系統科學系/碩士(2015年)

摘要

磁小體(Magnetosomes)在磁性細菌(magnetotactic bacteria)內之成長於生物科學及醫學仿生材料上有重要意義，如基因治療與癌症熱治療等。但迄今為止其成長機制仍未有定論。傳統上多以冷凍切片方式觀測磁性細菌與磁小體，因此難於觀測合成過程。本研究以臨場觀測磁小體成長過程為目標，據此設計一套實驗方式，其中包含(1)微機電製程設計應用於流體環境元件(Flow cell)開發與(2)穿透式電子顯微鏡(TEM)空錐暗場(Hollow Cone Dark Field)應用於液體環境拍攝。
於Flow cell因不同樣品之厚度需求，利用(1)金屬蒸鍍或(2)光阻塗佈設計墊高層(Spacer)控制液體厚度與空間，配合流體系統樣品台(liquid holder)，可提供養份與移除代謝物，並臨場觀察樣品變化。
為解決TEM拍攝生物樣品與生俱來的弱對比問題(來自樣品內的低原子序碳氫氧原素)因此利用TEM的兩組中間透鏡系統，將入射電子偏移離開中央軸，產生偏光空錐暗場(Tilt illumination Hollow Cone Dark Field)使影像對比增加。
現階段成功於流體系統中，觀察到磁小體受電子與水影響而溶解的過程，並利用空錐暗場於乾式環境拍攝磁性細菌鞭毛，鞭毛部分對比與明場相比大幅提升十一倍，於流體環境細菌影像對比亦提升四倍。

關鍵字

磁性細菌 ； 臨場流體穿透式電子顯微鏡

並列摘要

Growth of magnetosomes in magnetotactic bacteria is important in the biological sciences and application of medical biomimetic materials, such as gene therapy and cancer heat treatment. However, the growth mechanism is still inconclusive so far. Traditionally, magnetotactic bacteria and magnetosomes have previously been studied by cryo thin sections method, which is difficult to directly observe the synthesis process. In this study, we aim to observe the process of magnetosome synthesis, and according to this purpose to design a series of experiments, including (1) Application of MEMS process design on Flow cell development and (2) Application of Hollow cone dark field (HCDF) on photographing objects in liquid environment in TEM.
In Flow cell, we use (1) metal deposition, or (2) photoresist coating method to design different thickness spacers to control the thickness of the liquid and space. With combination of Flow cell and liquid holder, we can provide an open system for bacteria.
In order to solve the weak contrast of biological samples (arising from low atomic number elements of sample) , we use tilt illumination hollow cone dark field by adjusting two lens system to shift the incident electron from the central axis to increase the image contrast.

並列關鍵字

magnetotactic bacteria ； In-Situ Flow TEM

參考文獻

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