漸逝場激發之三維奈米解析度之驗證

近來單分子檢測法(single-molecule detection)是科學界熱門研究之一，其提供了直接觀測單分子在液態系統中的行為，並且定量測量出動態與動力學的參數。以往觀測法都是以整體平均測量的生化實驗為主，個別分子所反映的差異性常常會在巨觀測量下被忽略，因此直接觀測單分子行為，提供了科學家研究生物分子一個強而有力的工具。單分子螢光技術連接起結構生物學與生物化學兩個領域，為了進一步探討分子結構與功能之間的關係，觀測系統解析度須等於或小於分子等級。然而，在廣域的螢光顯微鏡技術受限於繞射極限(diffraction limit)，而螢光共振能量轉移法(FRET)則提供了當分子之間距離小於10nm時構形改變的訊息。所以在10nm與250nm之間，是目前大多數技術的瓶頸，但是許多令人感興趣的生物現象卻發生在這尺度下。論文的動機源於欲證明全反射螢光顯微術在三維解析度可達到奈米尺度，使得能觀察及量化大部份生物分子的行為，為此灰色地帶來一個新的契機。在橫向平面解析度方面，單一螢光分子形成的強度分佈圖形，經過高斯曲線擬合演算法運算後，其半高頻寬(FWHM)約為250nm，高斯曲線圖形的中心可代表為螢光分子的位置，當螢光分子位置產生變化時，可由圖形的中心變化判別出。在縱向解析度方面，本研究所採用的全反射螢光顯微術，其特性為在界面上能量分佈與縱向距離呈現指數遞減的關係，藉由螢光變化與穿隧深度值，即可換算出縱向距離的變化值。而為了驗證全反射螢光顯微術的三維解析度是否有達到奈米尺度，本研究結合全反射螢光顯微術與原子力顯微鏡，借用原子力顯微鏡的壓電掃描器，其具有奈米移動的解析度，經由比對兩個儀器所得到的資料，進一步驗證全反射螢光顯微術三維奈米等級之解析度。驗證全反射螢光顯微術具有能力觀測及量化細胞間或細胞內的分子訊息傳遞過程。利用全反射螢光顯微術搭配微機電製程製作的PDMS微流道，觀測綠色螢光蛋白質(GFP)於蓋玻片表面吸附的現象。有別於一般螢光顯微技術只能觀測橫向平面的動態行為，本研究成功地觀測到單一分子的三維動態行為。量子點具有尺寸大小決定發光波長，且只需單一雷射波長激發，發光時間可長達十小時的種種特性。在未來可運用量子點的特性搭配全反射螢光顯微術的優異解析度，將為細胞內與細胞間的單分子追蹤研究帶來一波新的革命。

關鍵字

漸逝場；解析度驗證；全反射螢光顯微術

並列摘要

Single-molecule detection (SMD) provides a way to observe and measure the behavior of single molecule directly. In the past ensemble measurements involving a large number of molecules, the differences between individual molecule are ignored. Therefore, SMD provides powerful tools to look into biomolecules. However, fluorescent microscopy is limited in its resolution by the Rayleigh criterion at ~250nm. On the other end of the size spectrum, fluorescence resonance energy transfer (FRET) provides a way to probe the events under 10nm. Distance measurements in the range of 10-250nm is the choke point of most techniques. Motivation of this thesis is originated from verifying the three-dimensional resolution in nanometer range of total internal reflection fluorescent microscopy (TIRFM) by using atomic force microscopy (AFM). Fluorescent beads are affixed to cantilever tip through chemical modification and use property of the piezoscanner which can resolve displacement in nanometer range. The tip is moved by piezoscanner and the images are collected by CCD simultaneously. With comparison of data of AFM and TIRFM, the resolution of TIRFM can be confirmed. To quantify horizontal displacement, this paper use Gaussian curve to fit the distribution of intensity profile of single fluorescent molecule. The center position of fluorescent molecule can correspond to the peak of fitting curve. The change of vertical distance can be determined from the change of intensity and penetration depth. To show that TIRFM is able to observe and quantify signal transduction in cell or between cells, we observe the phenomenon that GFP adsorb on the coverslip. Distinct from other fluorescent techniques that only observe the dynamic motion in the transverse plane, TIRFM is able to observe the three-dimensional motion of single molecule in real time. Quantum dot has a variety of features such as lifetime long as ten hours and only one wavelength for excitation. To make use of the characteristic of quantum dot and excellent resolution of TIRFM, it will be revolutionary of single-molecule detection in cell and between cells.

並列關鍵字

evanescent wave ； 3D resolution ； TIRFM

參考文獻

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[3] K. Kitamura, M. Tokunaga, A. H. Iwane and T. Yanagida,”A single myosin head moves along an actin filament with regular steps of 5.3 nanometers,” Nature,vol 397,pp.129,1999.

[4] Y. Harada, T. Funatsu, K. Murakami, Y. Nonoyama, A. Ishihama, and T. Yanagida, “Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time,” Biophys. J., vol. 76, pp. 709-715, 1999.

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漸逝場激發之三維奈米解析度之驗證

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