Single fluorescence imaging to observe the dipole radiation appearance between two same dye

2000年以後，德國Jörg Enderlein 發表一些文章關於單分子染料的失焦成像於CCD上的理論計算及利用染料cy5作的成像的實驗。觀察到染料分子的偶極在空間中三維取向不同，CCD上會有具有方向性圖形成像。於單分子單偶極成像技術中，兩個螢光染料分子的偶極的相對方向、及距離會影響於失焦時的影像的圖形（Pattern）及空間中相對強度的分布。Jörg Enderlein 在這方面已有相當完整的理論及實驗的驗證。本實驗中，更進一步利用文獻的結果；觀察廣場光學顯微鏡下的單分子雙偶極失焦時的圖形於CCD上的成像問題。不同於Jörg Enderlein的實驗，只針對單偶極的研究。本實驗更進一步觀察雙偶極的行為；並控制變因來觀察偶極作用的因素。 分子動力學的模擬（Molecular Dynamic simulation）方面，使用Gromacs Package 模擬染料分子於DNA在水溶液的平衡結構。並找出分子的局部的穩定結構，並使用ＭＭ-PBSA估算與其分子其他結構的自由能的差距。在sample選擇上使用線性的DNA分子並於５端的磷酸根上染料分子ＴＭＲ。利用ＤＮＡ的螺旋結構，作為距離、角度的控制。本來的目標，是希望利用分子動力學的模擬中染料分子於DNA在水溶液的距離、角度的分布。並由此推測空間上的染料相對位置，對應於顯微鏡下的失焦時影像的圖形。但因為實驗上的方便起見，並不是在水溶液中做實驗；故這部份的結果僅供參考。From 2000，Jörg Enderlein had some articles about experiment and theory in single molecular Cy5 defocus image in a sensitive CCD camera. Single fluorescence imaging method for direct determination of single-molecule orientations is presented that uses a wide-field epifluorescence microscope and a sensitive CCD camera. Jörg Enderlein had good results in theory and experiment. In this thesis，we use his result to observe the dipole radiation appearance between two same dye. In single molecule mage method , two dye’s dipole have different orientation to show different defocus pattern in CCD camera and pattern’s relative distribution. In Molecular Dynamic simulation，we use “Gromacs Package” to simulate the equilibrium structure of DNA which is labeled TMR in solution in water solution. We want to find the local minimus energy ,and MM_PBSA model’s free energy. Double strand linear DNA is the sample to do defocus image . We want to use DNA to control the distance and angle between two dye.But the condition in experiment is not solution. So, result of MD part is reference.目錄試委員會審定書 …………………………………………i言及謝誌 : .................................... ii文摘要： ..................................... iiibstract： ...................................... iv錄............................................. v目錄......................................... vii目錄.......................................... ix一章 序論...................................... 1.1 實驗動機.............................................1.2 單分子偵測方式.......................................................................................2.2.1 單分子偵測方法的優點...................................................................2.2.2 單分子偵測方式的基礎...................................................................3.2.3 廣場顯微術觀察成像.......................................................................4.3 單偶極成像概念及理論...............................................................................7.3.1 單偶極成像行為...............................................................................9.3.2 雙偶極的實驗目標.........................................................................13.3.3 雙偶極的實驗模型.........................................................................14.4 理論計算......................................................................................................16.4.1 Molecular Dynmaic 分子動力學.................................................16.4.2 離焦時成像圖形的模擬.................................................................20二章 實驗與方法................................ 21.1 樣品備制......................................................................................................21.1.1 DNA 事前的備制.............................................................................21.1.2 單分子樣品備制.............................................................................30.2 雙偶極離焦圖形之攝像與分析..................................................................33.2.1 雙偶極行為的概述.........................................................................38.2.2 雙偶極行為- Incoherent .............................................................42.2.3 雙偶極行為- Coherent .................................................................46.3 分子動力學計算.........................................................................................53三章 結果與討論................................ 54.1 雙偶極間輻射行為分析.............................................................................54.1.1 距離.................................................................................................54.1.1 雷射能量及相互間的方位探討......................................................55.1.3 偶極間角度及相位角......................................................................61.2 分子動力學結果.........................................................................................66.2.1 自由能計算.....................................................................................66.2.2 水相中結構模擬.............................................................................67四章 結論及未來展望............................ 70考文獻........................................ 72目錄ig 1-1 wide field 的聚焦示意圖.................................................................4ig 1-2 物鏡式廣場光學顯微鏡..................................................................5ig 1-3 偶極成像示意(Böhmer and Enderlein 2003) ..................................7ig 1-4 單偶極成像實驗及理論圖形..........................................................9ig 1.5 TMR 光偶極方向的示意................................................................10ig 1.6 TMR 螢光吸收放射光譜................................................................10ig 1.7 角度與成像 - 1..............................................................................11ig 1.8 角度與成像 - 2..............................................................................11ig 1-9 不同角度的單偶極理論成像 離焦 = 0.4 ~0.6μm......................12ig 1-10 單偶極實驗成像..........................................................................12ig 1-11 雙偶極成像的模擬......................................................................13ig 1-12 雙偶極實驗的模型......................................................................14ig 1-13 TMR & TAMRA 染料分子式及吸收放射光譜..........................15ig 1-14 15mer 起始結構與平衡時結構的選取...................................19ig 2-1 TMR 跟 linkage 的結構...............................................................24ig 2-2 標記反應的簡易機制示意............................................................25ig 2-3 HPLC 空白實驗..............................................................................27ig 2-4 HPLC 純染料..................................................................................27ig 2-5 HPLC_DNA 純化的圖譜...............................................................28ig 2-6 決定λ/4 的快慢軸........................................................................33ig 2-7 決定左右旋(http://www.newfocus.com) .......................................33ig 2-8 高斯分佈 曝光強度......................................................................34ig 2-9 曝光強度平滑修正........................................................................34ig 2-10 曝光強度圖w/cm2.......................................................................35ig 2-11 曝光無明顯明雙光子現象..........................................................35ig 2-12 單分子攝像組圖 - 1....................................................................37ig 2-11 單雙偶極截圖..............................................................................37ig 2-14 θ1 = 90 θ2 = 90 ΔΦ = 0 ~ 180 .....................................................38ig 2-15 θ1 = 90 θ2 = 70 ΔΦ = 0 ~ 180 .....................................................38ig 2 -16 兩個平躺偶極投影夾角變化造成的強度變化.........................40ig 2 -17 θ1 = 90 θ2 = 70 偶極投影夾角變化造成的強度變化..............40ig 2-18 單分子攝像組圖 - 2....................................................................42ig 2-19 單偶極理論實驗對照 – 1...........................................................43ig 2-20 雙偶極減去單偶極......................................................................44ig 2-21 單偶極理論實驗對照 – 2...........................................................44ig 2-22 雙偶極理論值<incoherent> ........................................................45ig 2-23 雙偶極分析理論實驗總結<incoherent> ....................................45ig 2-24 phase......................................................................46ig 2-25 單分子攝像組圖 – 2...................................................................47ig 2-26 單偶極理論實驗對照 – 3...........................................................48ig 2-27 雙偶極分析理論實驗總結<In phsae>........................................49ig 2-28 單分子攝像組圖 – 3...................................................................50ig 2-29 單偶極理論實驗對照 – 4...........................................................51ig 2-30 雙偶極分析理論實驗總結<Out of phsae>.................................52ig 3-1 數據於CCD 上的分布..................................................................55ig3-2 86bp ~18mw 能量對行為統計........................................................56ig3-3 86bp ~8mw 能量對行為統計..........................................................57ig 3-4 86bp Angle 統計............................................................................59ig 3-5 Phase angle......................................................................................60ig 3-8 15bp、29bp、39bp、86bp 長度的角度統計柱狀圖....................61ig 3-9 86bp - 圓極化光的旋光性對phase 的影響.................................63ig 3-10 39bp - 圓極化光的旋光性對phase 的影響...............................64ig 3-11 染料的stack free energy..............................................................66ig 3-12 MD 計算的距離分佈...................................................................67ig 3-12 MD 計算的角度距離分佈(0~180)...............................................68ig 3-12 MD 計算的角度距離分佈(0~90).................................................69ig 4-1 Stimulation 造成的波長窄化的現象.............................................71目錄able2.1 實驗DNA 的序列.......................................................................21able2.2 實驗中使用到的Buff .................................................................22able 2.3 離焦圖形攝像條件.....................................................................33able2.4 單分子攝像<Incoherent> ............................................................43able2.5 單分子攝像<coherent – In Phase>..............................................48able2.6 單分子攝像<coherent – Out of Phase>.......................................51able 3.1 不同長度DNA 行為..................................................................54able3-2 86bp 18mw 統計的資訊.............................................................58able3-3 86bp 8mw 統計資訊...................................................................58able 3-5 左右旋橢圓極化光對分佈的影響............................................65able 3-6 MD 的自由能計算結果(kcal/mol) .............................................66able 3-7 MD 計算結構的資訊..................................................................6