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研究生:許平諭
研究生(外文):Ping-Yu Hsu
論文名稱:雷射光引發之細胞膜表面形貌變化
論文名稱(外文):Laser-induced variations of cell membrane topography
指導教授:李超煌
指導教授(外文):Chaw-Huang Lee
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:90
中文關鍵詞:細胞膜動態雷射光毒性
外文關鍵詞:membrane dynamicslaserphototoxicity
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我們利用具有奈米縱向解析度的非干涉式廣視野光學測繪術(non-interferometric wide-field optical profiolometry, NIWOP)研究CL1-5人類肺腺癌細胞之細胞膜與雷射光間的交互作用。我們將雷射光源嵌入NIWOP系統,作為對細胞膜的刺激,在施予雷射光線照明後隨即以NIWOP記錄細胞膜表面形貌。細胞邊緣退縮及前伸的行為能夠分別被紫光(405 nm)和紅外光(1064 nm)所調控;照射時間與細胞反應的程度為一正比關係,且紅外光的照射時間必須比紫外光高出數百倍之多才能使細胞有所反應。我們發現聚焦雷射光的照射雖然使細胞邊緣有前進或退縮的反應,但是在細胞膜表面形貌的粗糙度與擾動性在照射前後並無明顯的差異;光線對細胞膜造成的擾動是非常微小的。另外我們也以螢光顯微術觀察了肌動蛋白濃度在雷射光刺激前後的分布變化,細胞膜緣退縮及前伸的行為分別伴隨了肌動蛋白濃度的減少與增加。此現象可能是由於紫光的光毒性會迅速破壞肌動蛋白骨架,而紅外光則能提升肌動蛋白單體聚合成細胞骨架的機率。我們確定光線幾乎沒有擾動細胞膜,但仍可刺激細胞使其濃度分布產生變化,其中確切的機制尚未明瞭,是還有待深入研究的。本篇論文將簡介數個與「雷射光—細胞交互作用」相關的研究工作,還有常被用以研究細胞膜表面形貌的工具。接著闡述NWOP的原理、光學系統架設以及它在細胞膜動態研究上的適用性與研究成果。然後詳細描述本研究的實驗步驟與數據分析方法,最後總結我們的研究結果。
We utilize non-interferometric wide-field optical profiolometry (NIWOP) that has axial resolution of tens of nanometers to study the interaction between laser light and the plasma membrane of CL1-5 human lung cancer cell. Two lasers are combined with NIWOP system to provide light stimulation for the plasma membrane. After laser irradiation we immediately record cell membrane topography by NIWOP. The retraction and protrusion of the edge of the cell can be modulated by violet light and infrared light, respectively. The response of the cell is proportional to the illumination time of the light. The illumination time of infrared light has to be a hundred time longer than that of the violet light in order to make significant stimulation for the cell. We found that although light irradiation makes cell edge retract or protrude, the roughness and fluctuation of cell membrane topography show no differences after light irradiation. The perturbation caused by light irradiation on the cell membrane topography may be negligible. Besides, the actin concemtration distribution is observed by fluorescence microscopy. The retraction and protrusion of the cell edge are accompanied by the decrease and the increase of actin concentration. This phenomenon may be due to cytotoxicity of violet light that rapidly destroys actin cytoskeleton and the ability of infrared light that promotes the actin monomer polymerization. We see that light irradiation stimulates change of actin concentration despite that it makes nonsense to the cell membrane. The actual mechanism is yet not fully understood and need to be further investigated. This thesis first introduces works that are related to “interactions between laser light and the cell” and the tools often used to study cell membrane topography. The principle of NIWOP and its optical setup will be explained, followed by a brief review in its applications in studying cell membrane dynamics. We then describe our experimental procedure and how the data are analyzed. In the end we make conclusions for the results.
第一章 研究簡介
1.1. 雷射光與細胞交互作用的研究...............................................................1-1
1.1.1. 雷射光微手術與生物分子的遞送.................................................. 1-1
1.1.2. 細胞內鈣離子的調控...................................................................... 1-3
1.1.3. 細胞生長導引................................................................................. 1-3
1.1.4. 紫光的影響..................................................................................... 1-5
1.2. 細胞膜形貌的研究工具...........................................................................1-5
1.2.1. 細胞膜的構造................................................................................. 1-6
1.2.2. 螢光顯微術..................................................................................... 1-7
1.2.3. 全反射螢光顯微術.......................................................................... 1-8
1.2.4. 原子力顯微術................................................................................. 1-9
1.2.5. 掃描式電子顯微術........................................................................ 1-10
1.3. 非干涉式廣視野光學測繪術的應用......................................................1-11
1.4. 實驗構想................................................................................................1-14
1.5. 參考文獻................................................................................................1-15
第二章 非干涉式廣視野光學測繪術簡介
2.1. 光學切片顯微術以及結構式照明...........................................................2-1
2.2. NIWOP與樣品表面形貌校正模型..........................................................2-7
2.3. 光學系統架設........................................................................................2-12
2.4. 參考文獻................................................................................................2-15
第三章 實驗方法及影像分析
3.1. 實驗方法..................................................................................................3-1
3.1.1. 細胞樣品備製................................................................................. 3-1
3.1.2. 雷射光照射..................................................................................... 3-3
3.1.3. 軸向反應曲線線性區歸一化斜率計算及NIWOP影像拍攝........... 3-4
3.1.4. 螢光影像光漂白測試...................................................................... 3-7
3.2. 分析方法..................................................................................................3-8
3.2.1. 細胞反應統計................................................................................. 3-8
3.2.2. 細胞膜表面形貌運算...................................................................... 3-9
3.2.3. 時變圖繪製................................................................................... 3-11
3.2.4. 表面粗糙度計算........................................................................... 3-12
v i
3.2.5. 表面擾動性計算........................................................................... 3-13
3.2.6. 觀察細胞骨架分佈........................................................................ 3-14
3.3. 簡短結語................................................................................................3-14
3.4. 參考文獻................................................................................................3-16
第四章 實驗結果與討論
4.1. 雷射引發之細胞膜表面形貌變化...........................................................4-1
4.1.1. 細胞對雷射光照射的反應.............................................................. 4-2
4.1.2. 參考區域選擇................................................................................. 4-4
4.1.3. 雷射光點下膜高度變化.................................................................. 4-6
4.1.4. 細胞膜粗糙度變化.......................................................................... 4-8
4.1.5. 細胞膜擾動性............................................................................... 4-10
4.1.6. 紅外光雷射引發的表面形貌變化................................................ 4-12
4.2. 細胞內肌動蛋白濃度變化.....................................................................4-14
4.2.1. 細胞退縮╱前伸時肌動蛋白濃度變化......................................... 4-14
4.2.2. 雷射光點處肌動蛋白分布............................................................ 4-16
4.3. 參考文獻................................................................................................4-19
第五章 總結與未來工作
5.1. 總結..........................................................................................................5-1
5.2. 未來工作..................................................................................................5-3
5.3. 本章參考文獻..........................................................................................5-5
附錄
6.1. 雷射—NIWOP系統用驅動程式..............................................................6-1
6.1.1. NIWOP laser plus Master driver...................................................... 6-1
6.1.2. NIWOP laser plus Slaver driver....................................................... 6-4
6.2. NIWOP資料處理介面..............................................................................6-4
6.3. 參考文獻................................................................................................6-10
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