雙光子螢光相干光譜系統發展與應用

Abstract

本論文研究發展雙光子螢光相干光譜(Two-Photon-based Fluorescence Correlation Spectroscopy)之系統與技術。螢光相干光譜是藉由量測螢光訊號的變動作相干擬合得分子的擴散係數、濃度以及分子間作用等等之訊息。擴散係數是量測有鍵結螢光分子的標的物在已知系統激發體積下做布朗運動所造成螢光訊號變動。進一步的利用不同的自相干曲線模型分析一種以上的分子交互作用下所擁有的擴散係數；倘若在分子有聚集的效應下，利用螢光相干技術可成功地自聚集情況下分辦出真正生物分子作用(例如:抗原與抗體結合)的訊號。另外，也發展利用描掃式螢光相干光譜技術量測固定於表面螢光分子，並藉由測量結果估算單位面積所含分子數目。 實驗結果顯示雙光子螢光相干光譜量測分子擴散係數的平均標準偏差量為0.0064。15nM的fluorescein混合不同甘油濃度(2.5%至40%)，其擴散係數從 246.173μm2/s下降至35.1626μm2/s。在抗原與抗體實驗，固定標的螢光分子的Goat Anti-Rabbit IgG的濃度與調變Goat Rabbit IgG的濃度，曲線擬合可以得到在分子結合構形的擴散係數16.9μm2/s達73.5%的最大結合率; 固定標定螢光分子的Goat Anti-Rabbit IgG的濃度與Goat Rabbit IgG的濃度，隨著時間點的不同做曲線擬合可以得到在分子結合構形的擴散係數34μm2/s 達60%的最大結合率。在量測表面固定螢光分子方面，在激發面積量測fluorescein平均數目為23.57個，因此，推算50μm × 50μm有208k個fluorescein。

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.