Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK.
Chemphyschem. 2011 Feb 25;12(3):500-9. doi: 10.1002/cphc.201000833. Epub 2010 Dec 29.
Molecular self-assembly is a defining feature of numerous biological functions and dysfunctions, ranging from basic cell signalling to diseases mediated by protein aggregation. There is current demand for novel experimental methods to study molecular self-assembly in live cells, and thereby in its physiological context. Förster resonance energy transfer (FRET) between fluorophores of a single type, known as homoFRET, permits noninvasive detection and quantification of molecular clusters in live cells. It can thus provide powerful insights into the molecular physiology of living systems and disease. HomoFRET is detected by measuring the loss of fluorescence anisotropy upon excitation with polarised light. This article reviews recent key developments in homoFRET fluorescence anisotropy imaging for the detection and quantification of molecular self-assembly reactions in biological systems. A summary is given of the current state-of-the-art and case studies are presented of successful implementations, highlighting technical aspects which have to be mastered to bridge the gap between proof-of-concept experiments and biological discoveries.
分子自组装是许多生物功能和功能障碍的一个决定性特征，从基本的细胞信号传递到由蛋白质聚集介导的疾病。目前需要新的实验方法来研究活细胞中的分子自组装，从而在其生理环境中进行研究。荧光团之间的Förster 共振能量转移（FRET），称为同型 FRET，允许在活细胞中非侵入性地检测和定量分子簇。因此，它可以为生命系统和疾病的分子生理学提供有力的见解。同型 FRET 通过测量用偏振光激发时荧光各向异性的损失来检测。本文综述了用于检测和定量生物系统中分子自组装反应的同型 FRET 荧光各向异性成像的最新关键进展。概述了当前的最新技术，并介绍了成功实施的案例研究，突出了必须掌握的技术方面，以弥合概念验证实验和生物学发现之间的差距。
