臺灣博碩士論文加值系統

本研究使用CMOS(Complementary Metal-Oxide-Semiconductor)模組搭配藍光LED進行細胞生長觀測，實驗中藍光LED激發CFSE染劑染色之小鼠巨噬細胞RAW264.7產生螢光，使用影像感測元件CMOS觀測細胞螢光表現，搭配LabView圖控程式，進行自動化控制影像擷取與激發光控制，形成多通道平行檢測，最後由NI VISION進行影像處理，統計細胞的成長以及螢光亮度變化，並且結合明視野(light field)，達成同時觀測染色螢光細胞之螢光表現及細胞在自然光下之生長情形，推算細胞倍增時間與細胞分裂指數，與血球計數器觀測的情形做比對；研究中小鼠巨噬細胞的倍增時間為24 hr，細胞分裂指數為66.59%，而血球計數器得到細胞倍增時間為17.2 hr，細胞分裂指數為68.75%，第一組至第四組細胞在倍增時間亮度減低分別為33.8%、41.3%、46.2%、50%，並且本系統取得放大效果40×至50×之螢光影像。

In this study, a real-time fluorescence detection system for cell growth analysis is proposed utilizing the CMOS (Complementary Metal-Oxide-Semiconductor) modules with blue LEDs. The RAW 264.7 murine macrophage cell labels with CFSE (Carboxyfluorescein succinimidyl ester) fluorescence dye and the blue LEDs are for cells fluorescence excitation for real time cell growth fluorescence monitoring. The CMOS modules and LEDs are controlled using custom-made LABVIEW code to capture real time cell growth fluorescence images automatically. The cell growth images are analysis by the NI Vision program.
In the proposed system, the cell proliferation was evaluated by doubling time and cells division index in the cells growth processes. In this study, the doubling time and cells division index of RAW264.7 cell are 24 hr, 66.59%. Compare to the traditional hemocytometer cells counter, the doubling time and cells division index of RAW264.7 cell are 17.2 hr, 68.75%. The fluorescence intensities decay in the doubling time are 33.8%, 41.3%, 46.2% and 50%, respectively. The CMOS module magnification rate is about 40 x to 50 x compare to the traditional microscope. As a result, the proposed system provides an ideal solution for cell proliferation real time fluorescence monitoring applications in the bio-medicine field.

摘要.............................................. I
Abstract..........................................II
誌謝..............................................IV
目錄.............................................. V
圖目錄......................................... VIII
表目錄.......................................... XII
簡寫表......................................... XIII
符號索引......................................... XV
第1章 緒論.........................................1
1.1 前言...........................................1
1.2 文獻回顧.......................................2
1.3研究動機與目的................................. 14
第2章 研究方法.................................... 15
2.1 簡介......................................... 15
2.2 螢光系統建立.................................. 16
2.2.1 影像擷取系統................................ 16
2.2.2 光學系統設計................................ 18
2.2.3 細胞培養皿載台與CMOS平台設計..................19
2.2.4 激發光源電源供應系統........................ 20
2.2.5 影像擷取與激發光源自動控制系統.................21
2.3 細胞培養過程...................................23
2.3.1 器材滅菌....................................23
2.3.2 調配培養基..................................25
2.3.3 培養液更換..................................25
2.3.4 細胞計數................................... 26
2.3.5 細胞染劑....................................27
2.4 影像後處理....................................28
2.4.1自然光細胞的影像處理..........................28
2.4.1螢光細胞的影像處理............................29
第3章 結果與討論..................................32
3.1 綜合討論......................................32
3.2 CMOS系統之驗證................................32
3.3 鏡頭可視範圍(FOV)與顯微鏡比較..................34
3.4 螢光檢測系統觀測小鼠巨噬細胞....................36
3.4.1無染螢光細胞成長情形..........................36
3.4.2 第一組螢光染劑巨噬細胞.......................39
3.4.3 第二組螢光染劑巨噬細胞.......................41
3.4.4 第三組螢光染劑巨噬細胞.......................43
3.4.5 第四組螢光染劑巨噬細胞.......................45
3.5 實驗結果數據分析..............................47
第4章 結論與未來展望..............................50
4.1 結論.........................................50
4.2 未來展望......................................52
參考文獻..........................................53
作者簡介..........................................56

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