北京生物医学工程

基于FRET荧光蛋白探针的活体定量分子影像方法探究

Quantitative in-vivo molecular imaging based on genetically-encoded FRET probes

作者：王颖奇唐吉刘晴白净刘晓冬

单位：清华大学医学院生物医学工程系(北京100084)

分类号：R318;Q631

出版年·卷·期（页码）：2016·35·6（566-570）

摘要：

目的荧光共振能量转移（fluorescent resonance energy transfer，FRET）探针由于自身仍存在诸多局限，其在活体成像领域尚未得到广泛应用。本文旨在解决FRET探针在活体定量分子影像应用中所存在的瓶颈问题,即如何利用FRET探针精准计算目标物浓度，以及如何基于FRET探针实现三维成像。方法基于探针和目标物结合时的化学反应平衡关系，提出一种新型分析方法，以精确定量待测物［钙调素(calmodulin, CaM)］的浓度。同时对于FRET探针的活体分子成像，结合荧光透射成像（3D-FMT）方法，建立了可进行三维时空动态FRET检测的平台系统，对FRET探针的测量结果（即待测物浓度）进行实时定量的三维重建。结果准确地定量了待测物CaM浓度，并得到了高质量的3D-FRET分布影像。结论提出FRET探针在活体成像应用领域两个关键问题的解决方案，为基因编码FRET探针向活体分子影像领域的推广奠定了重要的技术基础。

Objective There’re limitations in the application of Fluorescent resonance energy transfer (FRET) sensor in in-vivo molecular imaging and our research are trying to solve them. One of the bottleneck problems is how to quantify the binding equilibrium for the reaction between the probes and targets. The other problem is how to utilize FRET sensor to reconstruct 3-D images for temporal and spatial dynamics of the targeted molecules in the live body. Methods We take an established FRET sensor of calmodulin (CaM), BSCaMIQ, as the exemplar, by which we propose a new method to analyze the FRET ratio due to the binding between endogenous CaM and BSCaMIQ. We also utilize BSCaMIQ in a 3-D FMT system, by which 3-D FRET thus the CaM dynamics is attempted. Results we successfully quantified the concentration of CaM in live-cells and the 3-D FRET distribution of BSCaMIQ. Conclusions Our work provides the solutions for the two major bottleneck problems in applications of FRET probes to quantify targeted biomolecules, contributing to the technical foundations for future development of molecular imaging with FRET probes in vivo.

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