超解析螢光顯微鏡在生物研究領域已成為觀察細胞結構與動態過程的重要工具。由於其突破光學繞射極限的能力，超解析螢光顯微鏡能提供較傳統光學顯微鏡更高解析度的影像，從而揭示細胞內微小的結構變化。本研究的第一部分以粒線體為核心，作為細胞能量代謝中心，粒線體對環境變化敏感，且其動態變化與多種疾病相關。在本實驗中，我們測試了多種市售粒線體染料並分析其螢光特性，選擇適合雙染色的螢光染料，並設定每30秒拍攝一次，觀察粒線體在細胞暴露於缺氧環境及紫外光照射下的動態變化。這些環境條件會誘發粒線體的形態變化，可能會影響其功能與細胞健康。我們成功使用超解析徑向波動顯微術(Super-Resolution Radial Fluctuations, SRRF)觀察到在缺氧及紫外光照射的條件下，粒線體的形態由長條線狀轉變為甜甜圈狀或碎片狀，表明氧化壓力及環境改變對粒線體形態有顯著影響，可能進一步導致粒線體的功能失調或損傷。本研究的第二部分則聚焦於EBV（Epstein-Barr Virus）病毒顆粒的觀察。EBV與多種疾病相關，因此了解其進入細胞的機制至關重要。我們測試多種市售細胞膜染料並分析其螢光特性，發現MemGlow™ NR12A Membrane Polarity Probe在SRRF影像重建中效果最佳。隨後，我們使用MemGlow™ NR12A Membrane Polarity Probe搭配超音波震碎儀嘗試觀察EBV病毒顆粒。結果顯示經由PMA誘發後的HK1-EBV細胞經超音波震碎後，可以觀察到些許粒徑為100-300 nm的圓形顆粒，這些顆粒可能與EBV病毒顆粒相關，為研究EBV入侵細胞的動態機制提供新視角。

Super-resolution fluorescence microscopy has become an essential tool in biological research for observing cellular structures and dynamic processes. By surpassing the diffraction limit of conventional optical microscopy, super-resolution fluorescence microscopy provides higher-resolution images, revealing intricate structural changes within cells. The first part of this study focuses on mitochondria, which serve as the central hub of cellular energy metabolism and are highly sensitive to environmental changes. Mitochondrial dynamics are closely associated with various diseases. In our experiments, we tested multiple commercially available mitochondrial dyes and analyzed their fluorescence properties to select suitable fluorophores for dual staining. Time-lapse imaging was performed at 30-second intervals to observe mitochondrial dynamics under hypoxic conditions and ultraviolet (UV) irradiation. These environmental factors induce morphological changes in mitochondria, potentially affecting their function and cellular health. Using Super-Resolution Radial Fluctuations (SRRF) microscopy, we successfully visualized the transformation of mitochondria from elongated filamentous structures to ring-like or fragmented forms under hypoxia and UV exposure. This suggests that oxidative stress and environmental changes significantly impact mitochondrial morphology, which may further lead to functional impairment or damage. The second part of this study focuses on the observation of Epstein-Barr Virus (EBV) particles. EBV is associated with various diseases, making it crucial to understand its cellular entry mechanisms. We tested several commercially available membrane dyes and analyzed their fluorescence properties, identifying MemGlow™ NR12A Membrane Polarity Probe as the most effective for SRRF image reconstruction. Subsequently, we used MemGlow™ NR12A Membrane Polarity Probe in combination with ultrasonication to examine EBV particles. Our results showed that after PMA induction and ultrasonic disruption of HK1-EBV cells, small circular particles with diameters of 100–300 nm were observed. These particles may be associated with EBV, offering new insights into the dynamic mechanisms of EBV cell entry.

論文審定書 i
誌謝 ii
中文摘要 iv
Abstract v
目錄 vii
圖目錄 xi
表目錄 xv
第一章 超解析螢光顯微鏡介紹 1
1.1 緒論 1
1.2 傳統螢光顯微鏡 1
1.3 繞射極限 5
1.4 超解析螢光顯微鏡 9
1.5 單分子定位顯微鏡 11
1.6 超解析徑向波動顯微術 13
第二章 以超解析螢光顯微鏡觀察肌動蛋白絲、微管以及粒線體動態變化 18
2.1 肌動蛋白絲及微管介紹 18
2.2 粒線體介紹 19
2.2.1 粒線體構造 19
2.2.2 粒線體型態與疾病的關聯 21
2.2.3 粒線體常見染料 22
2.3 實驗動機 26
2.4 實驗試藥及溶液配製 26
2.4.1 實驗藥品 26
2.4.2 溶液配製 30
2.5 實驗設備 30
2.5.1 光學系統 30
2.5.2 ibidi系統 32
2.6 實驗方法 34
2.6.1 實驗用玻片製備 34
2.6.2 Huh7細胞繼代及培養於玻片 34
2.6.3 肌動蛋白絲及微管染色步驟 36
2.6.4 粒線體染色步驟 36
2.6.5 雙EMCCD系統之校正 39
2.6.6 影像及數據處理 39
2.7 結果與討論 40
2.7.1 SPY555-FastAct™濃度測試與ThunderSTORM影像重建 40
2.7.2 SPY650-FastAct™濃度測試與ThunderSTORM影像重建 42
2.7.3 ViaFluor® 488濃度測試與ThunderSTORM影像重建 44
2.7.4 ViaFluor® 647濃度測試與ThunderSTORM影像重建 46
2.7.5 MitoTracker™ Green FM probe濃度測試與SRRF影像重建 48
2.7.6 MitoTracker™ Deep red FM probe濃度測試與SRRF影像重建 50
2.7.7 NAO濃度測試與SRRF影像重建 52
2.7.8 JC-1濃度測試與SRRF影像重建 54
2.7.9 PKmito Deep Red濃度測試與SRRF影像重建 57
2.7.10 比較細胞骨架染料與粒線體染料之螢光穩定性 59
2.7.11 Resazurin對活細胞粒線體的染色成效 61
2.7.12 NAO與MitoTracker™ Deep Red FM probe對粒線體進行雙色染色 65
2.7.13 MitoTracker™ Green FM probe與PKmito Deep Red對粒線體進行雙色染色 69
2.7.14 最佳化拍攝間隔時間 73
2.7.15 缺氧環境對A549細胞粒線體的影響 77
2.7.16 UV照射對WS-1細胞的粒線體的影響 82
第三章 以超解析螢光顯微鏡觀察HK1-EBV細胞的病毒顆粒 89
3.1 Epstein-Barr病毒概述 89
3.2 研究動機 90
3.3 實驗試藥及溶液配製 91
3.3.1 實驗藥品 91
3.3.2 RPMI-1640培養液配製 94
3.3.3 含有50 ng/mL PMA的RPMI配製 94
3.3.4 含有1 mg/mL G418的RPMI配製 94
3.4 實驗設備 94
3.4.1 超音波震碎儀 94
3.4.2 即時聚合酶連鎖反應器 95
3.4.3 超解析螢光顯微鏡系統 95
3.4.4 ibid系統 95
3.5 實驗方法 95
3.5.1 HK1細胞及HK1-EBV細胞培養 95
3.5.2誘發EBV病毒 97
3.5.3篩選EBV陽性之HK1-EBV細胞 97
3.5.4細胞膜染色方法 99
3.5.5從HK1-EBV細胞中分離出EBV病毒顆粒 99
3.5.6 EBV病毒顆粒染色方法 99
3.5.7 影像和數據處理 101
3.6 結果與討論 102
3.6.1 CellMask™ Orange Plasma Membrane的濃度測試 102
3.6.2 CellMask™ Deep Red Plasma Membrane濃度測試 104
3.6.3 MemGlow™ NR4A Membrane Polarity Probe的濃度測試 106
3.6.4 MemGlow™ NR12A Membrane Polarity Probe的濃度測試 108
3.6.5 比較細胞膜染料之螢光穩定性 110
3.6.6 比較不同細胞膜染料的影像重建成效 111
3.6.7 HK1細胞之EBV誘發成效 113
3.6.8以細胞膜染料MemGlow™ NR12A Membrane Polarity Probe觀察EBV病毒顆粒 115
第四章 結論 118
參考文獻 120

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