設計與發展光切片顯微鏡術並應用其觀察活體秀麗隱桿線蟲

Design and Develop Light Sheet Microscopy for Live Canenorhabditis elegans

簡如萱(Ju-Hsuan Chien)

指導教授 ： 駱遠

國立臺灣大學/醫學院/醫療器材與醫學影像研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802345

摘要

即時顯微影像是研究生物解剖構造、機制和過程不可或缺的工具，其中傳統光學顯微鏡是最主要，也是最常用的工具，但礙於樣本的體積，傳統顯微鏡常無法取得合適的影像，因此有越來越多的影像技術被發展出來，例如共軛焦顯微鏡術，以配合研究上日益增高的需求。雷射共軛焦掃描顯微鏡需要以點對點的方式掃描標本因此掃描時間隨著樣本體積增大而延長，若掃描時間長，並不合適用來觀察活的標本，另外，由於雷射共軛焦掃描顯微鏡直接照明整個樣本，包括聚焦及失焦的部分，因此會增加光漂白和光毒性的可能。為了克服這個缺點，本研究設計及發展光切片顯微鏡，以得到快速的光切片影像，將有助於觀測有體積的活樣本。
光切片顯微鏡也被稱為層光螢光顯微術，是由兩條光路所構成，一條為照明光路，另一條為偵測光路，照明光路利用圓柱鏡將光單一方向的聚焦，使光束形成一層薄片，只有被薄片光照射或激發且同時位於偵測光路物鏡的焦點平面上的樣本才能成像，該層影像由二維偵測器(例如感光耦合原件或互補式金屬氧化物半導體)快速記錄下來，即可獲得即時非侵入性的光學切片影像。光切片顯微鏡具有低光漂白和低光毒性的優點，因此適合用來觀察活體樣本，並可進行影像的三度空間重組。
有很多論文及報告將光切片顯微鏡應用於斑馬魚、果蠅及秀麗隱桿線蟲的胚胎等，但卻一直無法使用光切片顯微鏡成功的觀察活體秀麗隱桿線蟲，主要是因為活體秀麗隱桿線蟲無法存於水中，而水是光切片顯微鏡的觀察環境。此外，在觀測過程中蟲體的抽動也是一大問題。要解決以上觀測的困境需要設計特別的容器來放置線蟲並配合適當的樣本處理方法。此篇論文，藉由自製的樣本容器及穩定的電動馬達平台，配合三種樣本處理方式，設計及發展出適合觀察活標本的光切片顯微鏡，成功的觀察活的秀麗隱桿線蟲的生殖系統，包含其活體內的卵及胚胎。
為了測試自製光切片顯微鏡的影像效果，我們比較同一個樣本在我們自製的影像系統與明視野顯微鏡及傳統螢光顯微鏡的影像，發現使用我們的系統能得到較高對比與較高解析度的影像，使我們得以觀察活體秀麗隱桿線蟲。此篇論文展示光切片顯微鏡的實驗設計與架構，並藉由觀察活體秀麗隱桿線蟲來測試系統的影像結果。

關鍵字

光學切片 ； 光切片顯微鏡 ； 層光螢光顯微術

並列摘要

Imaging a volumetric sample in real time is required to directly observe biological anatomy, mechanisms and process. There are a variety of microscopic imaging systems, such as confocal scanning microscopy, developed to provide fine optical sectioning capability. However, the main drawbacks of currently existing confocal systems are long aquisition time and severe photobleaching since confocal scans point by point and illuminates all the sample including in-focus and out-of-focus position. To overcome such problems and to obtain high-speed optical sectioning images, we have demonstrated light sheet microscopy (LSM) and shown real-time images which are benefit of thick live samples.
The LSM, which is also known as selective plane illuminaiton microscopy, consists of two light ways. One is the illumination part and the other is the detection part. The light source is modulated to a thin laminar light by a cylindrical lens which focuses light into a line. Only a sample at the detection objective lens, excited by the laser at the same time, can produce an image. The 2D image will be recorded rapidly by a detector, such as CCD or sCMOS, which obtains optical sectioning, non-invasive and real-time images. LSM is suitable for three-dimensional observation of a live sample because of its low photobleaching and phototoxicity.
Many papers have applied LSM for observing zebra fish, dorsophila, Canenorhabditis elegans (C. elegans) embryo and so on. However, there has only rare reports using LSM to image live C. elegans because the worm is hardly to be maintained in water, which is an important medium for the water-immersed objective used in LSM to image. Besides, the movement of the worm may worsen the situation. To conquer such problems, it is necessary to design a special sample holder to keep the worms and to develop an appropriate sample mounting methods. In this thesis, we have designed a light sheet microscope using a highly precise motor stage and developed an unique sample holder and three sample mounting methods for observing live thick samples. By using our LSM, we have successfully observed the reproductive system of a C. elegans, including oocytes and embryos.
To see if our LSM has higher image quality, we use our LSM, bright-field microscopy and conventional fluorescence microscopy to image the same sample of a C. elegans, and compared their image qualities. We found that through our LSM, we could obtain high contrast, high resolution and high-speed optical sectioning images. This thesis has experimentally presented and characterized LSM’s experimental design and setup. In addition, we demonstrated that this LSM was able to obtain three-dimensional images of a C. elegans in vivo.

並列關鍵字

Optical-sectioning microscopy ； light sheet microscopy ； selectve plane illumination microscopy

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