Development and Applications of Two-photon-based Fluorescence Correlation Spectroscopy
Date Issued
2007
Date
2007
Author(s)
Wang, Po-Kai
DOI
zh-TW
Abstract
In this thesis, we developed the fluorescence correlation spectroscopy (FCS) based on two-photon microscopy system to access physical parameters that give rise to fluctuations in fluorescence signal. As given excitation volume, the diffusion coefficient of molecules due to Brownian Movement into or out of excitation volume is determined. Furthermore, depended on autocorrelation analysis based on diffusion coefficients of molecules, the fraction of bound species from measured samples containing the dye labeled Goat Anti-Rabbit IgG and Goat Rabbit IgG can be separated. The scanning FCS based on autocorrelation with flow model was also applied to measure surface-bound fluorescent species. The mean standard deviation of diffusion coefficient measurement in our two-photon based system is about 0.064. The changes of diffusion coefficient with 15nM concentration solution of fluorescein are 246.173μm2/s to 35.1626μm2/s with different concentration solution of glycerol ranging from 2.5% to 40%. In the binding experiment between Goat Anti-Rabbit IgG and Goat Rabbit IgG, as the bound species of kinetics mode and end mode between with the diffusion coefficient of
34μm2/s and 16.9μm2/s, the maximal binding fraction is 60% and 73.5%, respectively. In the probing surface-bound fluorescent species experiment, we determined that N, the average number of molecules in the excitation volume, is 23.57. The total number of molecules of area scanned with 50μm × 50μm, which contains the equivalent of 8928 detection “areas”, is 208k.
Subjects
雙光子
螢光相干光譜
擴散係數
布朗運動
自相干曲線
two-photon microscopy
fluorescence correlation spectroscopy
diffusion coefficient
Brownian Movement
autocorrelation
Type
thesis
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