基于线性光谱分离的活细胞实时定量FRET显微成像技术研究

项目名称： 基于线性光谱分离的活细胞实时定量FRET显微成像技术研究

项目编号： No.81471699

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 陈同生

作者单位： 华南师范大学

项目金额： 80万元

中文摘要： 基于荧光蛋白(FPs)的荧光共振能量转移(FRET)定量检测技术主要受困于供体受体间的光谱串扰及其在细胞中的随机表达。申请人最近提出了将检测系统校正和光谱串扰修正与活细胞FRET探测分离的思想，极大地提高了活细胞中定量FRET显微成像与分析的速度和成功率。在此基础上，本项目拟重点研究多种基于线性光谱分离的活细胞快速定量FRET显微成像技术，并研发适合在活细胞中同时快速定量检测FRET效率和供体受体浓度比的宽场显微成像系统；然后利用研发技术和系统分别在正常细胞和凋亡细胞中研究BH3-only蛋白(Bid/Bim)、促凋亡蛋白(Bax)以及抗凋亡蛋白(Bcl-XL)之间相互作用的时空动态行为，揭示三类蛋白之间的精确分子调控机制。本项目建立的活细胞定量FRET显微成像技术适用于多种荧光成像系统和光谱仪，必将成为研究信号转导过程中生物大分子复合物的聚合、解离及其组分定性和活性时空变化的重要工具。

中文关键词： 荧光共振能量转移；线性光谱分离；定量检测；活细胞显微成像；信号转导

英文摘要： Fluorescent proteins (FPs)-based fluorescence resonance energy transfer (FRET) quantitation has two main issues:spectral crosstalks and random of FPs expression in cells. We recently proposed a novel and quantitative measurement of FRET that separates FRET quantitation into three independent processes: system calibration, spectral crosstalks corrections and live-cell FRET detection, largely improving the success rate of FRET quantitation inside live cell. This project aims to develop linear spectral unmixing-based real-time and quantitative FRET imaging microscopy to perform rapidly and real-time monitor the absolute FRET efficiency and donor-to-acceptor concentration ratio in single live cells. We will use these techniques to study the dynamics of the interactions among BH3-only proteins (Bid/Bim), proapoptotic proteins (Bax) and antiapoptotic proteins (Bcl-XL) in live healthy and apoptotic cells, respectively, to reveal the exact molecular mechanisms among the three types of proteins. The real-time and quantitative FRET microscopy developed here is sutable for a varity of fluorescence microscope and spectrophotometer, it thus must be an important tool for studying the spatio-temporal dynamics of aggregation, disassociation, component and activation of biomacromolecules in live cells.

英文关键词： Fluorescence resonance energy transfer;Linear Spectral unmixing;Quantitative mesurement;Live-cell microscopy;Signal transduction