서강대학교

초록/요약

Single-molecule experiment investigates the properties of individual molecules, and it has been proven to provide much more information than conventional ensemble measurements. Immobilizing single molecules on glass surface is one of the ways to study single molecule behavior. What we expect is that all immobilized molecules on glass surface should show the same properties irrespective of their positions and the observation time because they are the same molecules. In order to confirm whether (1) the molecules are homogenous and (2) the molecule does not change in time, we conducted single-molecule Fluorescence Resonance Energy Transfer (smFRET) measurement, one of the most popular single-molecule fluorescence measurements. Double-strand DNA with a single-strand overhang with Cy3 and Cy5 dye molecules attached (called donor and acceptor molecules, respectively) was used as our sample. By measuring the single molecule for a long time (temporal case) and many molecules at different positions for a short time (spatial case), standard deviations of these two FRET efficiencies vs. photon number changed differently, with the latter showing larger standard deviation for larger photon numbers. Because the photolumiscence decay lifetime of the acceptor (Cy5) molecule, studied by time-correlated single photon counting (TCSPC), is expected to probe molecule-by-molecule deviations and follow the deviations found from the smFRET efficiency experiments, we also carried out the measurements on the standard deviations of the photoluminescence decay lifetime of the Cy5 single molecules for both cases; temporal and spatial cases. The result showed that the acceptor lifetime distributions in both cases showed the same behavior as the FRET efficiency distributions in both cases. From these two experiments, the local environment (or position dependence of individual molecules) is found to be a major source of the inhomogeneity of the photophysical properties of single dye molecules.