Abstract
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.