全內反射螢光顯微鏡在單分子螢光影像的觀測實驗中是必要的工具。藉由這個工具，可以顯著降低來自焦點外的背景螢光雜訊，而激發雷射也會被完全阻擋。本實驗室建構一稜鏡式全內反射螢光顯微鏡系統以研究單分子螢光影像。
以532nm二極體雷射的消逝波為激發光源，可以觀察到Dragon Green螢光珠以及Cy3螢光分子。因為消逝波的螢光激發範圍很小(穿透深度大約200nm)，且其強度隨深度而指數衰減，這些特徵或許可以用於微陣列基因晶片系統實驗中，DNA分子雜合反應的動態研究。
目前雖然訊雜比不夠大而無法觀察到單分子螢光影像，小波分析仍然被應用在尋找光漂白事件的平均訊號強度變化—這個變化視為一個分子的螢光強度。
本研究的目標是以最佳化訊雜比的全內反射螢光顯微鏡系統研究DNA分子雜合反應之動態行為。未來將整合高解析度的雷射光鑷子系統和全內反射螢光顯微鏡系統在一個平台上，做為研究單分子問題的有力工具。

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a necessity in observing images of single fluorescence molecules. By using it the background fluorescence signal from out of focus fluorophores can be reduced significantly and also blocking the excitation laser light completely. I have set up a prism type TIRFM system to study single molecule fluorescence images.
Under the use of evanescent waves from a 532 nm diode laser for excitation, the Dragon Green fluorescent beads and Cy3 molecules were observed. Because the penetration depth of the evanescent wave is really short (i.e. about 200nm) and the intensity of the evanescent wave decreases exponentially with depth, these characters may be used to study the dynamics of DNA hybridization in microarray gene chip experiments.
Although the signal-to-noise ratio is not big enough to observe single molecule fluorescence, wavelet analysis was still applied looking for the mean intensity of photo bleach from single fluorescence molecules.
The goal is to reach to the best signal-to-noise ratio and to get single molecule fluorescence images. In this way the technique can then be used to analyze the dynamics of DNA hybridization. The high resolution optical tweezers and TIRFM system will be combined to study more biomedical problems in the future.

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