利用聚焦離子束電子束顯微鏡建立酵母菌3D圖像及加強圖像清晰度

Abstract

近年來電子顯微鏡的技術蓬勃發展，不僅使得生物領域的研究有了長足的進步，在檢測儀器與知識探索的相輔相成下，使我們更加的瞭解了生命與微觀世界的關係。本篇論文中我們將利用酵母菌作為觀察對象，聚焦離子束雙束顯微鏡不同於以往的掃描式電子顯微鏡與共聚焦顯微鏡，不僅能夠提供樣本表面的資訊亦不需將樣本製備成薄片，即能夠在離子束切割之下得到截面訊息，能夠觀察樣本內部的微結構，進而能夠建立整顆細胞的三維影像，對於研究細胞的整體資訊而言是相當有力的工具。傳統的化學處理步驟繁複且通常伴隨著劇毒藥品，經固定、脫水、包埋等程序，生物細胞的細微結構，往往在這些過程中可能流失或者變形，而與實際情形不同，而我們使用FIB Nova 600i搭配了冷凍系統，由於生物樣本是利用急速降溫冷凍，因此所能觀察到的構造應該是最接近真實的，而我們發現在新鮮細胞經由冷凍之後，切面即能觀察到細微結構。但在製作三維影像重建時，交界的清晰度是判斷次級構造非常重要的因素，因此我們做了一系列實驗，目前我們所能夠了解的，經過化學處理後，當FIB的切削電流愈小(1.5pA)主箱體的溫度愈低（＜-155ﾟC），所得的截面影像就愈清晰，除了對照FIB所拍攝之截面外，同時也比較重建出之新鮮細胞與傳統化學處理後的3D影像之差異，瞭解冷凍系統與聚焦離子束顯微鏡在生物結構的研究上所能夠提供的資訊以及其優勢。

In recent years, the technology of the electron microscope grows vigorously, not only make the research of the biological field have considerable progress, but develop in the instrument and increase with knowledge give us more understanding of life and relation of the microcosms. In this thesis, we utilized yeast as a target. Focused ion beam dual beam microscope is different from the scanning electron microscope and confocal microscope. It could not only offer the information on a surface of samples, but no need to prepare the sample into a flake. Namely, it can get sectional information which were cut by ion beam to observe the micro-structure within samples, and then can assemble those series pictures to three-dimensional images of the whole cell. It is a quite powerful tool to provide whole cells' information for studying morphology. The traditional chemical treatment is complicated and usually accompanied with poisonous medicines, after the process of fixation, dehydration and embedding, the slight structure of biological cells often might run off or deform in the course, and different from real situation. The tool we used, Focused ion beam microscope (FIB) Nova 600i, carries cryo-system, because the biological sample was frozen rapidly by liquid nitrogen; the structures that could be observed should most close to truth. We could observe the micro-structures in the section images of the frozen fresh yeast. While making three-dimensional image to rebuild whole cell, the factor for judging the secondary structure is very important, so we have made a succession of experiments to compare the difference of 3D images between fresh cell and traditional treated cell and to contrast their section images. Understand information and its advantage that cryo system and FIB can offer in the research of the biological structure.