The Single-molecule Fluorescence Polarization Detecting System

• Main Authors: Yu-Chia Chang, 張瑜家
• Other Authors: Wun-Shain Fann
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/96600796697395987965

碩士 === 國立臺灣大學 === 物理研究所 === 92 === A single-molecule fluorescence polarization (SMFP) detecting system enables us to investigate the time evolution of the dipole orientation of a chromophore. It has applications for a variety of studies such as determine the protein structure, detecting the rotational motion of a protein, characterizing the properties of a membrane, and so on. Within the framework of this thesis, we have successfully developed a SMFP detecting system by employing a polarization beamsplitter cube into a double-port scanning confocal microscope. The intensity ratio of the two linearly perpendicular polarization states emerging from the two ports of this system is 1:3 when a circular polarized light was incident into the system. Thin polystyrene films containing isolated DiIC18(3) small molecules which are commonly used in the membrane study consisting of only one chromophore were prepared to test the SMFP detecting system. As to the single-molecule detection, reducing background fluorescence and increasing signal to noise (S/N) ratio are two crucial issues. Cleaner implements used in the sample preparation not only can enlarge the S/N ratio but also can prevent quenching effect from taking place. With the effort on the removal of the contaminants introduced in the sample preparation processes we found out that polluted pipette may easily contaminate solvents in use, even when disposable tips are used and presumably the solvents wouldn’t have contact with the inner of the pipette. To examine the background intensity of the sample, it has been found out that the incident light with 1 �巰 for the raster scanning is more efficient than that with 33 nW as we did previously. After eliminating possible contaminations, the background intensity of the sample amounts to a value of three counts per 10ms which is very close to the dark count rate of the detecting system has been achieved.