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研究生： 黃建翔
Chien-Hsiang Huang
論文名稱： 使用結構照明顯微術觀察活體小鼠毛囊生長週期之變化
指導教授： 陳思妤
Szu-Yu Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 理學院 - 光電科學與工程學系
Department of Optics and Photonics
論文出版年： 2017
畢業學年度： 105
語文別： 中文
論文頁數： 78
中文關鍵詞： 結構照明顯微術 、小鼠 、毛囊生長週期
相關次數： 點閱：11  下載：0 
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    • 在生醫研究上使用光學顯微系統觀察生物樣本的時候，需要能夠快速並且穩定的獲取即時資訊以及提供樣本的完整性，而結構照明顯微術 (Structured Illumination Microscopy, SIM) 除了能夠提供光學切片的功能和有著不錯的影像對比度之外，還可以穩定並且快速的取得即時影像，因此成為了一種能夠提供生醫研究上觀察樣本的工具。
    以往使用光學顯微系統觀察毛囊的不同生長週期的型態時，需要將許多隻經過密蠟除毛後的小鼠在不同的毛囊生長週期時間上，將其背部一部份的皮膚切下並且犧牲掉小鼠，接下來再把皮膚毛囊幹細胞染上不同的螢光標記並做成樣本，藉此來觀察不同時間毛囊的生長週期狀態，但是這樣的實驗過程中沒有辦法觀察由同一隻小鼠毛囊生長周期變化的連續性，而且樣本染色的成功與否也會影響觀察的方向性和正確性，最後小鼠的犧牲也會造成小鼠資源浪費並且無法再次利用。
    本論文以綠螢光蛋白(Green Fluorescent Protein, GFP)基因轉殖的小鼠做為樣本，其背部經過密蠟除毛使所有毛囊同步進入毛囊生長的休止期，利用以數位微型投影機 (Digital Light Projector, DLP) 做為系統光源的結構造明顯微系統來進行活體小鼠的觀察，實驗將持續一整個毛囊生長週期的循環時間觀察毛囊生長周期過程中不同的型態樣貌。

    In biomedical research, when using microscope system to observe biological sample, it is necessary to have fast and stable data acquisition with completeness of samples. Structured illumination microscopy not only can provide optical sectioning and nice image contrast, but also capture image fast and stable. These advantages make structured illumination microscopy become a popularity-used system for biomedical research.
    When observing hair follicles with optical microscopy system in the past, different mice have to be waxed and sacrificed to get back skin samples at different stages of the hair growth cycle. The hair follicle stem cells of the samples should be strained with dye of fluorophores to enhance their contrast for observation. However, with these process, the observation can-not be focused on the same mouse in a complete growth cycle and the staining process plays a key role in the sample completeness and data accuracy.
    In this paper, green fluorescence protein (GFP) transgenic mice were used as the specimens. The mice were waxed to synchronize all hair follicles into anagen state of the growth cycle. By using the structured illumination microscopy system based on a digital light projector to observe the mice hair follicle in vivo. The observation will be last for a complete hair follicle growth cycle to long-term monitior the hair follicle stem cell morphology at different stages of the growth cycle.

    中文摘要.............................................................................................................. II Abstract.............................................................................................................. III 目錄.................................................................................................................... IV 圖目錄............................................................................................................... VII 表目錄.................................................................................................................. X 第一章 緒論...................................................................................................... 1 1-1研究目的與動機.......................................................................................... 1 1-2毛囊構造及生長週期................................................................................. 3 1-2-1皮膚構造............................................................................................... 3 1-2-2 毛髮及毛囊結構.................................................................................. 4 1-2-3 毛囊幹細胞.......................................................................................... 5 1-2-4 毛囊生長週期...................................................................................... 7 1-3毛囊研究文獻回顧.................................................................................... 10 第二章 基本理論............................................................................................... 12 2-1光學顯微術................................................................................................ 12 2-2具有光學切片能力的顯微術................................................................... 12 2-3結構照明顯微術....................................................................................... 16 2-3-1發展及基本觀念................................................................................. 16 2-3-2成像理論推導..................................................................................... 18 2-3-3延伸發展介紹..................................................................................... 20 第三章 實驗方法.............................................................................................. 22 3-1實驗架構................................................................................................... 22 3-1-1 結構照明顯微術系統架構................................................................ 22 3-1-2 數位微型投影機................................................................................ 24 3-1-3 二維光偵測器.................................................................................... 26 3-1-4 影像擷取控制.................................................................................... 26 3-1-5分光鏡選擇......................................................................................... 27 3-1-6系統解析度分析................................................................................. 28 3-2 條紋型式.................................................................................................. 32 3-3樣本準備................................................................................................... 35 3-3-1切片樣本............................................................................................. 35 3-3-2活體樣本............................................................................................. 36 第四章 實驗結果............................................................................................... 38 4-1切片樣本................................................................................................... 38 4-1-1以螢光標定CK14的切片樣本......................................................... 38 4-1-2以螢光標定CD34的切片樣本......................................................... 39 4-1-3以螢光標定CK14及CD34的Whole Mount 樣本...................... 41 4-2 GFP螢光小鼠毛囊生長之活體觀察...................................................... 43 4-2-1 第1到第8天................................................................................... 44 4-2-2 第11天到第17天............................................................................ 48 4-2-3 第20到第23天................................................................................ 53 4-3 毛囊整體比較.......................................................................................... 56 4-3-1 膚色.................................................................................................... 56 4-3-2 毛根及毛髮型態................................................................................ 57 4-3-3 毛根、髮幹以及毛囊孔洞大小........................................................ 60 第五章 結論....................................................................................................... 63 參考文獻............................................................................................................. 65

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