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研究生:林奕呈
研究生(外文):Yi-Cheng Lin
論文名稱:超高解析顯微術的發展-STED(受激放射耗乏)
論文名稱(外文):Development of high resolution microscopy –STED (Stimulated Emission Depletion)
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:63
中文關鍵詞:受激放射耗乏超高解析繞射極限
外文關鍵詞:STEDSuperresolutionDiffraction Limit
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隨著生醫研究之觀測尺度越來越小,對超高解析顯微術的需求也隨之提高,以往的光學顯微觀測技術,因受限於繞射極限的限制,而無法清楚看到分子間的交互作用,因此發展奈米級遠場光學顯微術對於生醫研究是很重要。其中,以德國Stefan W. Hell教授團隊發展的受激放射耗乏顯微術(Stimulated emission depletion )成果最為顯著,此技術突破了繞射極限的限制,能觀測到數奈米的解析度。

STED顯微術結合了受激放射(Stimulated emission)與可逆式飽和螢光躍遷(RESOLFT),使用一道雷射激發樣品發出螢光,並使用另一道與螢光放射波段不同的雷射,誘使原本位於螢光激發能階的電子以受激放射機制回到基態;並使此激發光強度大於飽和強度,使螢光激發能階出現耗乏(depletion)的現象,並根據第二道雷射的形狀,調整抑制自發螢光產生的區域。透過以上機制可使光學解析度大幅提升。
本實驗室將架設STED顯微系統,除了可觀測更微小的物體以及細胞間的交互作用外,並結合已發展成熟之時間解析技術FLIM/FRET,完成超高空間解析與時間解析的技術STED-FLIM系統的建立。

本篇論文將詳細介紹STED的原理,及目前架設的進度,且對使用樣品之特性加以討論,系統架設上分為光學系統以及掃描系統來做介紹。

目前已完成兩道雷射空間上位置的重合校正以及兩道雷射時間差的參數測試。但仍須克服系統像差對影像的影響,才能取得有 STED效應的良好影像。
The development of contemporary biomedical researches calls for noninvasive microscopic observation with resolution at molecule scale and sensitivity at single molecule level. However, conventional optical microscopy is confined by Abbe’s diffraction limit and the interaction between molecules is usually beyond observation. A far-field optical microscopy with nano-scale resolution is much sought after to meet the coming challenges. Among the super-resolution microscopy developments, the one on stimulated emission depletion developed by Dr. Stefan W. Hell of Max Planck Institute, Göttingen, Germany has proven as a robust and prospective technique. It can be combined with the time-correlated single photon counting (TCSPC) to achieve versatile purposes.

STED microsccpy effectively combines the stimulated emission and RESOLFT (Reversible Saturable Optical Linear Fluorescence Transition). An excitation laser is used to excite molecules to emit fluorescence. The other laser (the STED beam) at a wavelength in the longer part of fluorescence induces the excited fluorophores to transit from the excited state to the ground state via stimulated emission. Controlling the intensity and the profile of STED beam is the key to achieve resolution beyond diffraction limit.

In this work the STED microscopy is developed with aims to observe the interaction between molecules. TCSPC based FLIM/FRET module will also be integrated into the system to achieve nanoscale spatial and temporal resolution, the STED-FLIM system.

This thesis reviews the work of STED and reports the progress of our lab’s setup as wells the critical issues encountered in the present and future developments.
第一章 序論 1
第二章 原理 4
2.1 繞射極限 4
2.2 受激放射原理 7
2.3 可逆式飽和螢光躍遷 (Reversible Saturable Optical Linear Fluorescence Transition, RESOLFT) 9
2.4 受激放射耗乏效應 11
第三章 樣品之特性及製備 20
3.1 樣品特性 20
3.2 樣品配製 21
第四章 STED顯微系統架設及實驗方法 23
5.1 STED 顯微系統架設 23
4.1.1 光學系統 23
4.1.2 掃描成像系統 31
4.2 實驗方法 34
4.2.1 系統校正 34
4.2.2 實驗操作方法 36
第五章 實驗結果與討論 37
5.1 奈米金粒子校正影像 37
5.2 遭遇問題 38
第六章 未來應用 40
6-1. STED-FLIM 40
6-2. STED microscopy with a supercontinuum laser source 42
第七章 參考文獻 43
附錄一 46
附錄二 47
[1] Rittweger, E., K. Y. Han, S. E. Irvine, C. Eggeling, S. W. Hell (2009): "STED microscopy reveals crystal colour centres with nanometric resolution". Nature Photonics 3, 144-147

[2] Schmidt, R., C. A. Wurm, A. Punge, A. Egner, S. Jakobs, S. W. Hell (2009): "Mitochondrial Cristae Revealed with Focused Light". Nano Lett. 9 (6), 2508-2510.

[3] Willig, K. I., S. O. Rizzoli, V. Westphal, R. Jahn, S. W. Hell (2006): "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis". Nature 440 (7086): 935-939.

[4] Increasing the Resolution of Far-Field Fluorescence Light Microscopy by Point-Spread-Function Engineering, Stefan W. Hell

[5] L. Rayleigh, Scientific Papers, Vol. 4, Cambridge University Press, (1903), p. 235.

[6] Frank L. Pedrotti and S. J. Leno S. Pefrotti, 'Chapter 21: Laser Basics', Inroduction to Optics, second edition, P. 426-431

[7] Stefan W. Hell, ‘Toward fluorescence nanoscopy’ Nature Biotechnology, 21: 1347-1355

[8] Stefan W. Hell et. al., ‘Chapter 31: Nanoscale Resolution with Focused Light: STED and Other RESOLFT Microscopy Concepts’ Handbook of Biological Confocal Microscopy, third edition, SpringerScience+Businiss Media, New York, 2006

[9] Stefan W. Hell et. al., ‘Breaking the diffraction resolution limit by stimulated emission: Stimulated-emission-depletion fluorescence microscopy’ OPTICS LETTERS, 19: 780-782

[10] Stefan. W. Hell et. al., ‘Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission’ PNAS 97: 8206-8210

[11] Stefan W. Hell et. al., ‘Fluorescence depletion mechanism in super-resolving STED microscopy’ Chemical Physics Letters, 442: 483-487

[12] Stefan W. Hell et. al., ‘Resolution scaling in STED microscopy’ OPTICS EXPRESS, 16: 4154-4162

[13] Theory of Confocal Microscopy: Fluorescence Excitation and Emission Fundamentals
(http://www.olympusconfocal.com/theory/fluoroexciteemit.html)


[14] Gaussian, Hermite-Gaussian, and Laguerre-Gaussian beams: A primer, Francesco Pampaloni, Jörg Enderlein, arXiv:physics/0410021v1

[15] Tight focusing of vortex beams in presence of primary astigmatism, Rakesh Kumar Singh,1,* P. Senthilkumaran,1 and Kehar Singh1,2, J. Opt. Soc. Am. A/Vol. 26, No. 3/March 2009

[16] Wildanger, D., E. Rittweger, L. Kastrup, S. W. Hell (2008): "STED microscopy with a supercontinuum laser source". Opt. Expr. 16 (13), 9614 - 9621. )

[17] Gaussian, Hermite-Gaussian, and Laguerre-Gaussian beams: A primer, Francesco Pampaloni, Jörg Enderlein, arXiv:physics/0410021v1
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