Abstract
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.