Study of fluorescence spectroscopy based on decay time and correlation

Abstract

In this dissertation, I analyzed the characteristics of protein with the fluorescence spectroscopic methods such as the time-domain lifetime measurements and the fluorescence correlation spectroscopy (FCS) using the confocal microscope based on the time-correlated single photon counting (TCSPC). At first, I applied the FCS to the molecules in ultra-low concentration and simulated a statistical model of FCS based on a Poisson process. The statistical model confirmed that the relative mean square amplitude of the fluctuations is shown to be inversely proportional to the average number of molecules, when the signal-to-noise ratio and measurement time are carefully controlled. Next, the lifetime characteristics of the enhanced cyan fluorescence protein (ECFP) were investigated. The ECFP lifetimes can be changed in relation to the pH value and integration times. The protonation is the main cause of the pH dependence of ECFP lifetime. The average lifetime variations according to the relatively short integration time can be overcome by the acquisition of the sufficient photon counts. However, there can be trade-off between the accuracy of the lifetime estimation and the information loss. Lastly, the conformational change of the maltose binding protein (MBP) was investigated by the fluorescence decay time method. The ECFP and the enhanced yellow fluorescent protein (EYFP) were fused to the MBP as a donor and an acceptor, respectively. The donor lifetime decreases when MBP undergoes conformational change, leading to closure of the two domains where the ECFP and the EYFP were fused. The donor lifetime shortening showed dependence on the maltose concentration. The auto correlation amplitude and the brightness were suggested as another indicator for the detection of FRET.