荧光相关光谱单分子检测系统的研究

Investigation on the Setup of Fluorescence Correlation Spectroscopy for Single Molecule Detection

基于激光共焦构型,构建了一套高灵敏度和高稳定性的荧光相关光谱单分子检测系统。采用高数值孔径物镜使激光束高度聚焦,并用同一物镜收集样品的荧光,单光子计数器用于实时记录荧光信号。优化系统保持物镜焦点、针孔以及单光子计数器光敏区高度同轴。采用60×1.2(NA)的水浸物镜及直径为160μm的针孔得到的检测系统结构参数为ω0和z0分别为0.42μm和1.33μm,照射微区的体积为1.3fL。单个RhodamineGreen分子可获得13倍的信噪比。用该系统获得了小分子和生物大分子(DNA片段和蛋白质)的高质量荧光相关图谱,成功实现了单个分子的检测。

Fluorescence correlation spectroscopy (FCS) is a novel technique for single molecule detection with wide potential applications in chemistry and life science. In this study a sensitive and stable FCS setup for single molecule detection was developed based on a confocal mode. The highly focused laser beam was obtained by use of the objective with high numeric aperture, and the fluctuant fluorescence signals were collected by a sensitive single photon counting module. The key factor for getting the successful FCS was to align the pinhole with both the objective focus and the active area of the single photon counting module. Utilizing the water immersion objective with 60 x 1.2 (NA) and the pinhole with 160 mu m, the defined radial and axial radius of this home-built setup were 0.42 mu m and 1.33 mu m, respectively. The illuminated volume was 1.3 fL, and the ratio of signal-to-noise for single Rhodamine Green molecule was 13. This FCS setup was successfully used for the analysis of individual small molecules and bio-macromolecules (protein and deoxyribonucleic acid fragments).