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研究生:莊建芹
研究生(外文):Chien-Chin Chuang
論文名稱:光譜解析螢光生命期顯微術的發展
論文名稱(外文):Development of spectrally resolved fluorescence lifetime imaging microscopy
指導教授:高甫仁
指導教授(外文):Fu-Jen Kao
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:65
中文關鍵詞:螢光生命週期顯微鏡光譜解析生命週期螢光生命週期顯微術共振能量轉移
外文關鍵詞:FLIMspectrally-resolvedlifetimeFLIMFRET
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本論文有兩個部分。第一部份探討量子點和奈米金桿當作FRET施者與受者之可行性。首先藉由靜電作用力將帶負電的量子點(施者)及帶正電的奈米金桿(受者)連接在一起。利用FRET限制條件中的光譜重疊越多,FRET效應會越強;以及FRET發生時施者與受者的距離需在1~10奈米內,這兩個方向來證明。在光譜重疊部分,將放射光譜位於605奈米的量子點,分別加入吸收峰值為650、700和750奈米的奈米金桿做FLIM測量。證明了生命週期會隨著光譜重疊的越多而減少。另外,在奈米金桿的外圍分別包覆厚度為10與15奈米的二氧化矽。證實當施者與受者距離超過10奈米後,沒有FRET效應。以上兩項數據驗證了當量子點和奈米金桿連接在一起時,量子點會藉由共振將能量轉移給奈米金桿。
第二部份結合單光儀與FLIM系統,發展一套"光譜解析螢光生命週期顯微平台",用來研究螢光分子之放射光譜與生命週期間的關係。發現在化學螢光染劑R6G與螢光分子eGFP之放射光譜各波段下生命週期數值的分佈相當一致。另外在FRET發生時,施者放射光譜中各波段下的FRET效益皆差不多。最後發現低量子效益光譜中,靠近放射光譜峰值處的生命週期分佈較一致。
The studies in this thesis are twofold. In the first part, we are investigating the fluorescence resonant energy transfer (FRET) between quantum dots and gold nanorods as the donor and the acceptor, respectively. The FRET are examined by varying the spectral overlapping and the distance between the donors and the acceptors. The emission peak of the quantum dots is at 605 nm. Gold nanorods with absorption peaks located at 650, 700, and 750 nm are attached to the quantum dots. The FLIM measurements show that the magnitude of lifetime decrement, indicating the extent of FRET, increases with the spectral overlapping. Te gold nanorods are also coated with polymer with thickness of 10 and 15 nm to vary the interacting distance between the quantum dots and the gold nanorods. The FRET efficiency is found to decrease to 1% when the distance is increased to beyond 10nm. These two measurements show that the quantum dot can transfer the energy to the gold nanorods by resonance.
Additionally, we have successfully developed a spectrally resolved module for the fluorescence lifetime imaging microscopy platform. It is used to correlate the lifetime and the fluorescence spectrum. As somewhat expected, we found that the lifetime and the FRET efficiency are similar at the various parts of the emission spectrum.
謝誌 i
摘要: ii
Abstract: iii
目錄: iv
圖目錄: v
表目錄: vii
第一章 緒論 1
第一節 螢光(fluorescence)之基本特性 1
第二節 奈米結構粒子 6
第三節 研究背景 9
第四節 研究目的 11
參考文獻: 12
第二章 實驗原理簡介 15
第一節 螢光生命週期影像顯微術 15
第二節 螢光能量共振轉移 17
參考文獻: 20
第三章 樣品資訊 21
第一節 Rhodamine 6G 21
第二節 增強綠色螢光蛋白 (enhanced green fluorescence protein, eGFP) 22
第三節 奈米金屬粒子 22
第四節 膠原蛋白 (collagen) 24
參考文獻: 26
第四章 實驗架設 27
第一節 時間解析 28
第二節 光譜解析 34
第三節 光譜解析螢光生命週期顯微術 36
參考文獻: 37
第五章 量子點與奈米金粒子之螢光共振能量轉移 38
第一節 實驗結果 38
第二節 討論 42
第三節 結論 44
第六章 光譜解析螢光生命週期顯微術 45
第一節 實驗結果 45
第二節 討論 54
第三節 結論 57
第一章:
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[12] Peter, M. & Ameer-Beg, S., Imaging molecular interactions by multiphoton FLIM. Biology of the Cell 96 (3), 231-236 (2004).
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[14] Wallrabe, H. & Periasamy, A., Imaging protein molecules using FRET and FLIM microscopy. Current opinion in biotechnology 16 (1), 19-27 (2005).
[15] Haluska, C. et al., Combining Fluorescence Lifetime and Polarization Microscopy to Discriminate Phase Separated Domains in Giant Unilamellar Vesicles. Biophysical Journal 95 (12), 5737-5747 (2008).
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[21] Michalet, X. et al., Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307 (5709), 538-544 (2005).
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[30] Li, X., Qian, J., Jiang, L., & He, S., Fluorescence quenching of quantum dots by gold nanorods and its application to DNA detection. Applied Physics Letters 94, 063111 (2009).
[31] Peter, M. et al., Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophysical Journal 88 (2), 1224-1237 (2005).
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第二章:
[1] Lakowicz, J. & Masters, B., Principles of fluorescence spectroscopy. Journal of Biomedical Optics 13, 029901 (2008).
[2] Becker, W., Advanced time-correlated single photon counting techniques. (Springer, 2005).
[3] Semwogerere, D. & Weeks, E., Confocal microscopy. Encyclopedia of Biomaterials and Biomedical Engineering. G. Wnek, and G. Bowlin, editors. Taylor and Francis, New York (2005).
[4] Denk, W., Strickler, J., & Webb, W., Two-photon laser scanning fluorescence microscopy. Science 248 (4951), 73-76 (1990).
[5] Suhling, K., French, P., & Phillips, D., Time-resolved fluorescence microscopy. Photochemical & Photobiological Sciences 4 (1), 13-22 (2005).
[6] Forster, T., Zwischenmolekulare energiewanderung und fluoreszenz. Annalen der Physik 437 (1948).
[7] Biskup, C. et al., Multi-dimensional fluorescence lifetime and FRET measurements. Microscopy Research and Technique 70 (5), 442–451 (2007).
[8] dos Remedios, C. & Moens, P., Fluorescence resonance energy transfer spectroscopy is a reliable" ruler" for measuring structural changes in proteins Dispelling the problem of the unknown orientation factor. Journal of Structural Biology 115 (2), 175-185 (1995).

第三章:
http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=R4127%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC
[2] Nikoobakht, B. & El-Sayed, M., Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chem. Mater 15 (10), 1957-1962 (2003).
[3] Fratzl, P., Gupta, H., Paschalis, E., & Roschger, P., Structure and mechanical quality of the collagen–mineral nano-composite in bone. Journal of Materials Chemistry 14 (14), 2115-2123 (2004).
[4] https://chempolymerproject.wikispaces.com/file/view/collagen_%28alpha_chain%29.jpg
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