利用葉綠體研究FIB-SEM削切技術

Abstract

在電子顯微鏡技術的快速發展下，利用電子顯微鏡來觀測細胞組織中的微 結構也成為熱門的研究領域。而在此領域中，利用穿透式電子顯微鏡(TEM)來觀測細胞切面已經廣泛的被利用，且在切面解析度上已取得良好的成果。在此篇論文中，我們將採用目前較常運用在半導體切面檢測之FIB-SEM(聚焦離子束雙束顯微鏡)系統來削切生物樣品。FIB-SEM系統的最大特色在於其不僅能提供樣本表面之訊息，能夠選擇我們計畫觀測之細胞樣品來削切觀測，不需將樣本製備成薄片，即能夠在離子束削切之下得到截面訊息，對於研究細胞的內部資訊與結構而言是相當有力的工具。 在以往之FIB-SEM觀測過程中，實驗者由於半導體技術之影響，習慣於表面濺鍍一層金屬來增加導電度以防止電荷累積的現象。然而，這個步驟在生物樣本上，卻有可能造成熱破壞而影響樣本的狀態。因此，在此篇論文中，我們不採用濺鍍金屬之方式，而是在未鍍金屬的狀態下進行削切與觀察的動作。也由於沒有金屬層的保護，對於樣品的處理、基板的選擇、FIB之參數選擇與特殊處理就顯得格外重要。因此我們做了一系列的實驗，在樣品處理方面，我們採用OTOTO處理法增加樣品導電度、低角度斜切減少應力、高導電度基板與接地金屬結構加上絕緣氧化層阻隔背像散射電子、正確的離子束削切參數使切面簾幕效應減少，我們將能夠有效的改善切面二次電子影像解析度，使得原本無法觀測之切面影像變得清晰可見，解決了FIB-SEM長久以來為人詬病之問題，也使得此技術能夠有效的應用於生物觀測上。

It is very important to perform electron microscopy to explore the biological field, especially while the ultra-fine structure of the bio-samples is of concern. Among them, the transmission electron microscope (TEM) has played a critical role on the resolution of a cross section image. Instead, in this thesis, we alternatively deployed focused-ion-beam (FIB) technique incorporated with a scanning electron microscope (SEM) to mill biological samples. The FIB-SEM system has been widely used in the semiconductor industry. Also, in the biological categories, such a double beam system can not only offer the milled surface information, but also provide an opportunity to choose the location and the field of milling in real-time. Furthermore, it is unnecessary to do the plastic embedding and thin slicing while we required for TEM. With FIB-SEM to mill and observe semiconductors, it is usual to sputter a metal layer on the surface of the samples, to improve conductivity and to avoid charge accumulation. The maneuver is also inherited to use on the biological samples’ surface in the previous reports. However, such a metal sputtering may not be beneficial to the biological samples. In this thesis, we don’t use platinum sputtering; instead we mill the biological samples directly. Due to the elimination of metal sputtering, it becomes more crucial that samples preparation, milling substrate, and FIB controlling parameters must be meticulously adjusted and monitored according to the milling results. We hereby developed a series of methods such as OTOTO method, grounded substrates, low milled angles, ion beam recipes to improve the secondary electron image resolution of the milled surface of chloroplasts. With this novel method, FIB-SEM system may turn into a powerful tool for the biological investigation for the first time in its history.