臺灣博碩士論文加值系統

隨著科技的進步，近三十年來低溫穿透式電子顯微鏡技術崛起，無論硬體或軟體均有極其大之發展及貢獻，尤其是在生物上之應用，已可用於一些蛋白質結構之解析，對於生物體結構之辨識，無疑是一項利器。另外，低溫電顯技術之樣品前製製備技術，將含水之生物體以快速冷凍的方法(速凍法)，使樣品封存於無序冰中，最重要的一環是能夠保有生物體樣品之原始形貌，對瞭解生物樣品無疑是一大進步。
利用速凍法製成之冰樣品在電顯觀測時，由於電子束穿透樣品，會使樣品遭到電子束的破壞，可能產生氣體分子而形成氣泡；另高能量電子束也會造成冰融化而使樣品型貌改變，亦可能會直接造成樣品損傷。為解決此問題，以低電子劑量條件來做為觀察生物樣品，是操作低溫電顯的基本要求。但即使是以低電子劑量條件來拍攝低溫電顯樣品，為求得更多之影像訊息，可能把拍攝時間拉長；而拍攝時間一旦拉長，累積劑量仍會造成樣品損傷。過去文獻曾有討論利用糖類分子添加入低溫電顯樣品溶液中，可能對電顯樣品有保護作用，可降低電子束造成之損傷。本論文實驗即使想探討利用海藻糖來做為低溫電顯樣品保護劑之功效。
本實驗是以溶液中之微脂粒作為電顯樣品來進行研究，添加不同濃度之海藻糖溶液，利用速凍法製成無序冰低溫樣品，在低溫環境下改變不同之電子劑量拍攝條件，探討海藻糖添加物對延緩電子束對樣品之損傷的效用。實驗結果顯示，添加海藻糖作為低溫電顯樣品保護劑，確實可有效延遲電子輻射對樣品損傷時間。此方法應用在低溫電鏡觀測上應有其實用性。

In the past 30 years, cryo-transmission electron microscopy (cryo-EM) technology has been developed in many aspects for the applications on observing biological and medical specimens. Cryo-EM has been proven to be able to analyze some protein structures Cryo-EM allows users to quick-freeze the bio-samples in vitrified ice, observe the sample under the near-native condition, thus is a powerful tool to understand the samples in the native liquid phase. However, during cryo-EM operation, the high energy electron beam could quickly damage the ice sample. Therefore low-dosage imaging technique is usually used for cryo-EM. Even so, taking an image for a long exposure time could easily result in damaging on the icy bio-samples. Adding some sugar into bio-sample solution before TEM sample preparation was known to preserve the bio specimen better against the damage caused by the electron beam. This work aims for studying the effect of trehalose addition on preserving bio-samples under cryo-EM operation. Liposome was used as the bio-specimens for this study. In this work, various concentrations of trehalose solution were mixed with liposome solutions and made into cryo-EM specimens, then were taken images under different conditions of electron dosages used. The results show that trehalose addition is in fact effective in slowing the electron beam damaging occurring time on the cryo-EM specimen. This finding could be useful for practical applications of cryo-EM operation.

中文摘要.......................................i
英文摘要.......................................iii
誌謝..........................................v
目錄..........................................vi
表目錄........................................viii
圖目錄........................................ix
第一章 緒論....................................1
1.1 研究背景.............................1
1.2 研究方向及目的........................3
第二章 理論基礎與文獻回顧.........................4
2.1 低溫電子顯微鏡技術.....................4
2.2 生物顯微樣品製備技術...................5
2.3 電顯樣品冷凍技術之冷凍劑................6
2.4 微脂粒...............................7
2.5 生物電顯樣品保護劑.....................8
2.5.1 海藻糖.............................8
第三章 實驗方法.................................12
3.1 藥品及儀器...........................12
3.2 實驗流程與製備方法.....................14
3.2.1 製備海藻糖溶液與不同海藻糖濃度之含微脂粒混合
糖溶液.............................14
3.2.2 樣品製備與鑑定流程...................17
3.3 Cro-TEM冷凍樣品......................18
3.3.1 冷凍樣品之製備流程...................18
3.3.2 多孔洞支撐碳網銅網前處理..............19
3.3.3 速凍法樣品之製備.....................19
第四章 結果與討論................................30
4.1 冰晶.................................30
4.2 海藻糖在無序冰中之微脂粒形貌.............31
4.3 控制電子束在拍照前對樣品照射時間~0秒，電子破壞最低的狀況下微脂粒在海藻糖不同濃度無序冰中之形貌.......32
4.4 電子劑量1.8e-/Å2條件下拍攝之微脂粒形貌及電子束
照射時間破壞性探討......................34
4.5 電子劑量3.8e-/Å2條件下拍攝之微脂粒形貌及電子束
照射時間破壞性探討......................41
4.6 以海藻糖作為低溫電顯保護劑添加在微脂粒溶液中之濃
度探討................................47
4.8 實驗結果...............................48
第五章 結論......................................50
參考文獻.........................................51

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