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研究生:桐生文佳
研究生(外文):Fumika Kiryu
論文名稱:聚焦雷射誘發細胞骨架蛋白微管形成之動態
論文名稱(外文):Microtubule formation dynamics of cytoskeletal proteins by focused laser beam
指導教授:杉山輝樹
指導教授(外文):Teruki Sugiyama
口試委員:許 馨云吉川洋史
口試委員(外文):Hsu, Hsin-YunHiroshi Y Yoshikawa
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:英文
論文頁數:39
中文關鍵詞:微管雷射雷射捕陷動態
外文關鍵詞:MicrotubuleLaserLaser trappingDynamics
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透過聚焦雷射誘導的蛋白質纖維在空間與時間上的形成動態已有所研究。此篇論文中,以在活細胞內的微管、驅動及細胞骨架蛋白形成的動態纖維結構之混合溶液作為樣品。當一連續波長雷射(λ = 1064 nm)聚焦在溶液的液氣介面時,會誘發微米大小的蛋白質聚集體且纖維呈徑向排列。蛋白質聚集體的大小隨雷射瓦數及輻射時間而增長。縱然聚焦雷射在溶液裡面或者在固液介面也會有蛋白質聚集體形成,雷射聚焦在液氣介面時卻無法形成清晰的纖維結構。再者,在三磷酸腺苷(adenosine triphosphate, ATP)—微管及驅動蛋白纖維網絡動態運動的化學能的提供下,能產生巨大的聚集體。這些結果表明聚焦雷射在氣液介面不會發生明顯的蛋白質變性下,可以產生動態的蛋白質纖維網絡。
為了近一步了解因聚焦雷射而形成的纖維網絡動態,我們將透過螢光及偏光顯微鏡來監測溶液情況。螢光影像清晰表明出雷射聚焦處附近的蛋白質濃度在輻照雷射開始下瞬間提升。另外,偏光顯微鏡顯示蛋白質聚集體明顯的光學各向異性,確保了在雷射誘發下高度有序(非隨機)形成的纖維結構。
有趣的是,聚集體中的纖維結構在線性偏振光束下比在圓性偏振光束下更傾向有序的狀態,這證實了光電場對於有序的纖維形成有著至關重要的作用。再者,當雷射聚焦在溶液中的金薄膜上時,因電漿加溫所引起顯著的溫度提升,使較低的雷射功率可誘導出蛋白質聚集體。由於我們已知微管蛋白可在高濃度及高溫下更有效形成纖維,因此我們可得出結論—聚焦型雷射引起的光電效應及光熱效應均應考慮進蛋白質纖維網絡的形成機制。此實驗所得出的這些發現將提供光學操縱對控制蛋白質自組織系統有更新的見解。
Spatiotemporal dynamics of network formation of protein fibers by focused laser beam was studied. In this work, mixed solutions of tubulin and kinesin, cytoskeletal proteins that form dynamic fiber structures in living cells, were used as samples. The focused irradiation of air-liquid interfaces with a continuous-wave laser beam (λ = 1064 nm) induced the formation of micrometer-sized protein aggregates where fibers were radially oriented. The size of the protein aggregates was increased with laser power and irradiation time. Although protein aggregates were also formed by focusing the laser beam inside solutions and at the solid-liquid interface, clear fibrous structures observed upon the irradiation at air-solution interfaces were not created. Also, larger aggregates were obtained in the presence of adenosine triphosphate (ATP), which is chemical energy for the dynamic motion of the fiber networks made by tubulin and kinesin. These results indicate that the focused laser irradiation at air-liquid interfaces can produce dynamic protein fiber networks without significant protein denaturation.
To further understand the formation dynamics of the fiber network by a focused laser beam, the solution was also monitored by fluorescence and polarized microscopies. The fluorescence images revealed that protein concentration around the laser focus was increased immediately after the onset of the laser irradiation. Also, polarized microscopy showed apparent optical anisotropy of the protein aggregates, ensuring that highly ordered (not random) fibrous structures were formed by the laser irradiation. Intriguingly, the fiber structures in the aggregates tend to be more ordered upon the linearly polarized beam compared to the circularly polarized beam, supporting that the photoelectric fields play an essential role in the ordered fiber formation. Furthermore, When the laser was focused to a gold film in solutions that causes significant temperature elevation via plasmonic heating, the formation of the protein aggregates can be induced with lower laser power. Since tubulin is known to form fibers more effectively at higher concentrations and temperatures, we conclude that both photoelectric and photothermal effects of the focused laser beam should be involved in the underlying mechanism of the formation of protein fiber networks. These findings obtained in this work will provide new insights into the control of protein self-organization systems by optical manipulation.
中文摘要 II
Abstract III
Acknowledgment V
Contents VII
List of Figures IX
List of Tables XII
Chapter 1. Introduction 1
1.1 Laser trapping 1
1.1.1 History of optical trapping 1
1.1.2 Theoretical treatment of laser trapping 1
1.1.3 Laser trapping for biomaterial use 4
1.2 Microtubule as cell structure 4
1.3 In vitro application of cytoskeleton 5
1.4 Aim of this research 7
Chapter 2. Experimentals 9
2.1 Solution setup 9
2.1.1 Tubulin purification 9
2.1.2 Sample preparation for laser irradiation 10
2.2 Container preparation 12
2.2.1 Hydrophilization of glass 12
2.2.2 Chamber structure 12
2.3 Optical setup 13
Chapter 3. Results and Discussion 15
3.1 Formation dynamics of microtubule by laser irradiation 15
3.2 Mechanism and dynamics of aggregate formation 15
3.2.1 Adsorption of protein at air-solution surface 16
3.2.2 Marangoni flow 17
3.2.3 Possible mechanism: Radially-shape aggregates formation 17
3.3 Competitivity of temperature and concentration effect 18
3.3.1 Concentration dependence of aggregates 18
3.4 Aggregates observation by transmission electron microscope 19
3.5 Formation dynamics of microtubule with chemical energy 21
3.6 Formation dynamics of microtubule in each laser properties 26
3.6.1 Focal position dependence 26
3.6.1.1 Possible mechanism: Aggregates formation at air-water-solid interface 27
3.6.1.2 Possible mechanism: Aggregates formation in solution 28
3.6.1.3 Possible mechanism: Aggregates formation at solid-liquid interface 28
3.6.2 Laser power dependence 28
3.6.3 Polarization dependence (linear/circular) 30
3.7 Plasmonic heating 31
Chapter 4. Conclusion 36
References 37
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