染料分子在溶液中呈現因溶劑分子隨機碰撞產生的布朗運動行為，因此在顯微鏡聚焦範圍內外運動，將造成螢光訊號強度起伏。本論文以含稀薄染料分子（Rhodamine B）的甲醇溶液（濃度 至 間），分別在共焦顯微架構與全反射式架構下，探討溶液中染料分子在雷射光激發下不同參數之影響螢光訊號強度起伏之分析。
我們用5個主要的參數去做討論其螢光的特性，分別為:第一在共焦架構與全反射式架構間的差異，其兩者在雷射聚焦有效區域與強度分布的變化有明顯的不同，其中共焦架構有效聚焦區域為一個橢球狀的光強度分佈圖區域，在全反射式架構中其聚焦的有效區域只有界面的100-200 nm可以有足夠的光強度具有激發螢光分子的能力，全反射式架構的聚焦區域遠小於共焦架構的聚焦區域，所以在背景雜訊與螢光訊號中會有較好的區分，也適合較高濃度樣品。；第二為不同濃度下的差異，可了解在單個或多於一個或沒有分子存在時在溶液間進行布朗運動造成螢光強度變動差別與其光學特性上的探討；第三為聚焦位置改變下的差異，其會受雷射聚焦有效區域在溶液不同位置時所表現不同的光學性質，亦可了解在我們的架構中有效聚焦位置的範圍；第四為不同的激發強度；第五為螢光相關光譜的探討。
我們的結果指出在全反射式架構下呈現較低的背景，此時選擇溶液的濃度適合在較高的濃度。此外，在有效聚焦區域中的染料分子小於一時，在螢光相關光譜(FCS)縱軸部分的高度値不在與1/N一定比例有關，而是與N有關，其中N為在染料分子的濃度。

The motion of dye molecules in the solution is highly influenced by the Brownian motion caused by the stochastic collisions with the solvents, and it results the fluorescence intensity fluctuation. The thesis study the fluorescence intensity fluctuation of dilute dye molecule (Rhodamine B) in methanol solution ( - ), under confocal and total internal reflection (TIR) microscopy configurations.
Five parameters are used to probe the fluorescence characteristics: (1) the difference between confocal and the TIR configurations. The configuration influences the laser focusing area and consequently the intensity distribution. The effective focusing area in confocal configuration is an ellipsoid shape, while that of TIR configuration is a disk shape around the interface with depth 100-200 nm. It results the TIR configuration less background and higher concentration capability. (2) concentration. We control the concentration from much less than one molecule to more than one molecule in the effective focusing area, and we observe the change of burst intensity distribution. (3) the focus position. By changing the focusing position, we study the effective focusing region changes. (4) excited intensity, and (5) fluorescence correlation spectroscopy (FCS).
Our results indicate that TIR configuration exhibits lower background, and is suitable to higher concentration solution. In addition, when the dye concentration in the focusing area is much less than 1, the FCS amplitude is no longer follow 1/N, but rather be proportional to N, where N is the concentration.

摘要......................................................................................................................1
致謝......................................................................................................................4
目次......................................................................................................................5
圖表目錄..............................................................................................................7
第一章：緒論......................................................................................................10
1.1背景...........................................................................................................10
1.2研究動機...................................................................................................11
第二章：原理......................................................................................................12
2.1 螢光分子簡介.........................................................................................12
2.2 螢光簡介.................................................................................................13
2.3 全反射螢光顯微鏡之原理及架設.........................................................15
2.3.1全反射螢光顯微術....................................................................15
2.3.2 全反射式理論............................................................................15
2.3.3 漸逝波(evanescent wave)...........................................................18
2.3.4 螢光強度之計算........................................................................21
2.3.5 中間層........................................................................................22

2.4 螢光相關光譜偵測.................................................................................23
第三章 單分子螢光實驗步驟與儀器簡介......................................................28
3.1 單分子螢光實驗儀器架構.....................................................................28
3.2 實驗步驟.................................................................................................29
第四章 實驗結果與討論..................................................................................31
4.0 實驗數據探討之前言.............................................................................31
4.1 實驗背景與訊號的探討.........................................................................35
4.2 共焦架構與全反射式架構的比較.........................................................39
4.3 不同聚焦位置的比較.............................................................................42
4.4 Rhodamine B螢光分子在不同濃度下的光學特性…............................42
4.5 Rhodamine B螢光分子在不同激發光強度的光學特性........................44
第五章 結論與未來工作..................................................................................47
5.1 結論.........................................................................................................47
5.2 未來展望.................................................................................................47
第六章 參考資料..............................................................................................49

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