衣藻細胞於濕式電子顯微鏡中之觀測

Observation of Chlamydomonas reinhardtii in the Environmental Wet Electron Microscope

陳佑庭(YU-TING CHEN)

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/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00463

摘要

本研究之目的，在於利用電子顯微鏡取代解析度不足之光學顯微鏡作為觀察的工具，並配合微機電技術所製作的微型濕式腔體元件，將潮濕、液態的樣品封存於元件中藉此與電鏡的真空環境作隔絕，如此使得我們能夠在具高解析度電子顯微鏡下觀察於液體中的原生態細胞。
在背向散射電子成像模式的SEM中觀察上述樣品的影像，可發現液體(水)順利保留於元件中，然而存在於液體中的衣藻樣品卻無法在影像上觀察到，經過先將衣藻固定於觀測薄膜上之實驗後，發現水加入會對於影像的對比產生嚴重的負面影響，並從文獻中發現因為水與細胞在影像中細胞之於液體的對比只有2.7%而無法呈現，為解決此問題，使用對細胞相對毒性較低的甘油取代水可能是一解決的方式。
而在TEM的影像中所能帶給我們的資訊相當有限，這可能是由於覆蓋於細胞上方的液體使得電子束直徑擴展，以及細胞厚度過厚(≈5μm)使電子束產生大量散射行為，因而無法獲得良好的解析度，有鑒於此我們藉由反應式離子蝕刻與纖維素分解酶兩種方式將細胞進行薄化的動作，然而在效果仍不顯著的情況下或許利用外力將細胞壓扁將是一可嘗試的作法。

關鍵字

環境穿透式電子顯微鏡 ； 濕室環境腔體 ； 活體細胞觀測 ； 衣藻

並列摘要

Abstract

In this paper, the wet-cell which was manufactured by MEMS processes is used for isolating the hydrated, liquid sample from the vacuum environmental of electron microscope. Thus we can observe the native state cell under the electron microscopes which have much higher resolution than optical microscope.
Through the images of the above sample which obtained by a BSE mode SEM, we can confirm the liquid is sealed in the wet-cell. However, the image of Chlamydomonas reinhardtii cannot be observed and after the test of attaching cell to the thin film, we understand that the contrast between water and cell are too low to appear. A solution for solving this problem is that replace the water by glycerine to enhance the contrast of image.
On the other hand, the images which acquired from TEM bring limited information. This may result from the electron beam broadening by liquid, besides thick cell cause large amount scattering therefore high resolution can’t be achieved. On that account we use reactive-ion etching and cellulase enzymes to thin the cell, however the effect is not apparent. By means of mechanical force to compress the cell may be a method to improve this problem.

參考文獻

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