臺灣博碩士論文加值系統

細胞為生命之最小單位，個體的特殊性皆來自細胞的影響。幹細胞為身體各組織的原始細胞，擁有分化為其他不同細胞的能力，究竟是何種反應發號施令仍舊是個謎題，若能對單一幹細胞進行研究觀察，將有助於了解其中奧秘，因而科學家推動了單一細胞的研究。微機電系統技術可製作多種與生物分子尺寸相符的微小元件，有助於操控單一細胞，故結合微機電技術與生物醫學將利於單一細胞之研究。
本研究目的於建立一套單一細胞操控、培養與觀測之系統，研究方法可分為細胞分離與運送、培養與觀測分析。在細胞分離與運送方面，透過微機電製程輔助，對單一細胞進行操控。在細胞培養上，使用微型細胞培養室，將細胞培養於微量井晶片上，此晶片主要為提供單一細胞獨立成長環境，並利用晶片上的結構可對細胞進行培養液之替換或注入不同化學物質以刺激細胞，觀察細胞成長反應之現象。觀測系統則以全反射式螢光顯微鏡與CytovivaTM150等之顯微系統為主，即時觀測取得生物影像，並透過電腦軟體進行影像分析。
整合以上技術後，即需要生物知識來協助實驗研究，以正確解讀生物訊息，如細胞形變、蛋白質之運作等，進一步對單一細胞研究作更深入的探討。

Cell is the smallest, biologically functional system unit in lives, and every feature in organisms is constituted by cells. The most original cells in humans are stem cells which possess the remarkable potential to be developed into various cell types in body. The mechanism remains unknown such that a study of single stem cell is becoming a highly potential approach to unveil its activities. By taking the advantages in MEMS technique of comparable size to a cell, the manufacturing, manipulation and investigation for a single cell have been made in this work.
The objective of the study is to develop a microsystem of single cell for manipulation, culturing, and real-time investigation. In manipulation for a single cell, a micro-pipette and micro-well device has been micromanufactured in order for isolation and transportation of a single cell. In cell culturing, a single cell is trapped and cultivated within a micro-well chip inside the micro-incubation culture system. The chip provides an isolated environment for a single cell in which the device is allowed to be injected with chemicals and biomarkers to stimulate the single cell. Moreover, in cell observation, the real-time investigation has been made simultaneously by both the total internal reflection fluorescence microscopy and CytovivaTM150. The images can be obtained and analyzed with the image processing software such that a great deal of bio-information for a single cell can be achieved. With such a system, the single cell morphology and protein-protein interaction can then be potentially realized. The well-integarted cell microsystem has been made for potential study for a single stem cell or its associated interaction, differentiation and communication to specific cells.

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 x
第1章 緒論 1
1-1前言 1
1-2研究動機 2
1-3文獻探討 4
1-3-1 細胞實驗室(The lab in a cell) 4
1-3-2 單一細胞操控 6
1-3-3微小化全自動分析系統(Micro total analysis system, μ-TAS) 15
1-4研究方法 17
1-5論文架構 18
第2章 實驗原理 19
2-1 單一細胞操控 19
2-2 全反射式螢光顯微鏡之原理與架設 20
2-2-1 全反射現象之原理 21
2-2-2 漸逝波 22
2-2-3 光場架設之種類 24
2-2-4 螢光物質 26
2-3 CytovivaTM150之原理與架設 31
2-3-1 光學原理 31
2-3-2 解析度之增強 34
2-3-3 光學架設 36
第3章 實驗步驟 38
3-1 微量滴管與微量井之製作 38
3-1-1 晶片設計 39
3-1-2晶片製程 43
3-1-3 晶片整合 52
3-2 全反射式螢光顯微鏡之光場架設與校準 54
3-3 CytovivaTM150之改裝於倒立式顯微鏡與校準 58
3-4 細胞培養環境與系統架設 60
3-5 系統整合與流程 64
第4章 實驗結果與討論 69
4-1 細胞種類與前處理 69
4-2 群體細胞之培養與觀測 70
4-2-1 全反射式螢光顯微鏡於細胞觀測之優點 77
4-2-2 CytovivaTM150於細胞觀測之優點 78
4-3 單一細胞操控 79
4-3-1 單一細胞分離 79
4-3-3 單一細胞運送與固定 83
4-4 單一細胞培養與觀察 86
第5章 結論與未來展望 94
5-1結論 94
5-2未來展望 95
參考文獻 97

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