利用全反射螢光顯微術進行單分子偵測

Single Molecule Detection by Total Internal Reflection Fluorescence Microscopy

鄭淑雅(Shu-Ya Cheng)

指導教授 ： 蘇志明

國立臺灣大學/理學院/化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02098

摘要

單分子偵測因可呈現動態行為與動力參數的真實波動與分布、可進行蛋白質反應事件操縱與反應之定量分析、且反應毋須同步，故為生物分子研究之重要且實用的方法。而所謂單分子螢光顯微術，乃是利用適當波長的雷射，激發所選擇的螢光標識分子，透過對螢光標識分子的偵測與觀測，追蹤目標分子的動態行為。

　　本篇論文主要是關於建立一套稜鏡式全反射顯微螢光顯微術系統，以進行單分子研究。全反射顯微術成功的關鍵，在於有效地降低背景螢光值。全反射在界面所建立的漸逝場，其強度為入射光能量的數倍，並隨離界面的距離增加而呈指數性衰減，其滲透深度頗為狹窄，僅侷限於界面附近0.1微米的帶狀區域，故能選擇性激發臨近界面的螢光分子。漸逝場中的螢光標記分子經激發所放出的螢光訊號，經高數值孔徑的物鏡收集後，分別以電荷耦合元件（CCD）及雪崩式光二極體（APD）偵測。電荷耦合元件的優點在於空間的解析與多個單分子資訊的同時擷取，雪崩式光二極體的優點在於時間解析度，可用於單頻道或雙頻道的光子計數，以進行時間系列的解析。

　　針對ATP合成酶，本實驗選擇CyDye作為螢光標記分子，包括Cy3及Cy5，並採用532nm綠光雷射作為Cy3的激發光源。將濃度在10~100pM等級的CyDye洋菜溶液，旋鍍製成含水達99%的洋菜膠體薄膜以進行系統測試。測試的結果，無論是CCD影像分析，或是APD光子計數量測，均顯示本實驗所建立的稜鏡式全反射顯微螢光顯微術系統，確實具有進行單分子研究的能力，其中最主要的證據係Cy3單分子單一步驟光淬滅的行為。本實驗的背景螢光值僅每秒約20個單光子，訊號背景比值平均在13左右，最高達20，且咸信仍有提升空間。在本實驗系統的觀測下，Cy3螢光單分子在發生光淬滅之前，約可產生100,000∼1,000,000個光電子訊號。

　　單分子實驗系統確立後，未來將可以生物馬達為主角，進行單分子動態行為分析與動力學研究，包括以Cy3配合Cy3，進行螢光共振能量傳遞（Fluorescence Resonance Energy Transfer）之實驗，以期透過化學觀點，探討生物馬達的工作。

關鍵字

旋鍍 ； 光子計數 ； 漸逝場 ； ATP合成酶全反射顯微螢光顯微術 ； 單分子偵測

並列摘要

Single molecule detection is a powerful and practical approach to explore bio-molecules, because it provides information about actual fluctuation and distribution of dynamic and kinetic parameters, enables relationship between inputs and outputs of events of proteins reaction to be qualified, and removes the need for synchronization. We use a laser of suitable wavelength to excite the selected fluorescent-labeled molecules, and then address the dynamic behaviors of target molecules by analysis and observation of the labeled molecules. This is namely single molecule fluorescence microscopy.

　　This work is mainly about the construction of a Prism-type Total Internal Reflection Fluorescence Microscopy system for the purpose of single molecule studies. The key of TIRFM is to reduce the background fluorescence effectively. When the light is totally reflected in the interface, the evanescent field is generated. The evanescent field is several times the intensity of the incident light, and diminishes exponentially with the distance form the interface. The penetration depth is quite narrow, around 100nm. Fluorescent-labeled molecules, which happen to be in this region, are excited selectively and emit fluorescence. The signal is collected by a high numerical aperture objective, and detected by CCD and APD. CCD is able to provide spatial information and extract statistics on numbers of individual molecules, while APD offers adequate temporal resolution. The signal pulses from one or double channels were counted and analyzed with time series.

　　The reporters that are chosen for ATP synthase are CyDye, including Cy3 and Cy5. We use a green laser (wavelength = 532nm) to pump the Cy3. The substrates are spin-coated with Cy3 in agarose solution of 99% water as a test specimen, with concentrations in the range of 10~100pM. Both the CCD visualization and the APD photon counting measurements demonstrate that the TIRFM does work and is capable of probing single molecules. The background fluorescence value in our experiments goes around 20 photo-electrons/sec and the SBR averages 13, 20 at most, and still could be enhanced. Before photo-bleaching, about 100,000∼1,000,000 photo-electrons were detected from each Cy3 molecule by APD.

　　Since the single molecule detection system has been built up successfully, we will carry on the investigation into the dynamic behaviors of bio-molecules and the study of the enzyme kinetic, such as fluorescence resonance energy transfer between Cy3 and Cy5, so as to elucidate the underlying mechanism of bio-motors from a chemist’s point of view.

並列關鍵字

SMD ； Cy3 ； spin coating ； photon counting ； TIRFM ； evanescent field ； single molecule detection ； ATP synthase ； Total Internal Reflection Fluorescence Microscopy

參考文獻

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