為了達成單分子螢光的偵測，所以藉由全內反射螢光顯微鏡做螢光激發，來大幅減低背景雜訊，增加單一螢光的可見性。本實驗室利用商用的物鏡式全內反射螢光顯微鏡結合Dual-View與EMCCD來即時觀測單一螢光分子。
將螢光分子Cy3標記在單股DNA分子上，並藉由抗原抗體結合力，把DNA分子固定在玻片表面，再利用消逝波激發螢光，來觀測單一螢光分子光漂白的現象。
使用物鏡式全內反射螢光顯微激發系統，已經可以觀測到單一螢光分子光漂白的現象。為了得到最佳的訊雜比，分別使用三種不同的數據分析方法，其中以取螢光分子區域周圍25個像素平均值的方法，能夠得到最佳的訊雜比。藉由將雷射功率降低，觀察到單一螢光分子強度降低，以及螢光分子被光漂白的時間拉長，而螢光分子被光漂白的時間拉長，對單分子研究是很重要的。
利用雙股DNA分子分別標記上螢光分子Cy3與Cy5，希望能夠觀測到單分子螢光共振能量轉移現象。未來將會把物鏡式全內反射螢光顯微激發系統結合微流道，來觀測單分子動態行為，並整合高解析度雷射光鉗系統，來解決更多感興趣的生物研究。

In order to detect single molecule fluorescence signal, so I used total internal reflection fluorescence microscopy (TIRFM) to excite the fluorescent molecule. This setup can decrease much noise, and increase the chance of detection single fluorescent signal. Our laboratory used commercial TIRFM to combine with Dual-View and EMCCD, reached to single fluorescent molecule detection.
The sample of my experiment is single strand DNA labeled Cy3. The single strand DNA are fixed to the cover slip surface by antibody-antigen binding force. Then I used the evanescent wave by total internal reflection to excite the fluorescence, and to observe the phenomenon of single fluorescent molecule photobleaching.
In my TIRFM system, we already detected single fluorescent molecule photobleaching. In order to obtain better signal to noise ratio (SNR), I used three methods to analyze my experimental data. The method of mean value of 25 pixels in the fluorescence spot can obtain better SNR. By reducing the laser power, I detected the time of fluorescence photobleaching is increased, and the intensity of single fluorescent molecule is lowered. To enhance the time of fluorescent lifetime is important in the single molecule experiments.
I hope to observe the phenomenon of fluorescence resonance energy transfer (FRET) by using the double strands DNA labeled Cy3 and Cy5. The TIRFM system will combine with flow chamber to reach single molecule dynamical researches, and be integrated to high resolution laser tweezers system to study more biological programs in the future.

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