Live-cell FLIM-FRET using a commercially available system.
Live-cell FLIM-FRET using a commercially available system.
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DOI：
10.1016/bs.mcb.2020.02.002
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发表时间：
2020
影响因子：
--
通讯作者：
Maresca TJ
中科院分区：
文献类型：
--
作者：
Castellani CM;Torres-Ocampo AP;Breffke J;White AB;Chambers JJ;Stratton MM;Maresca TJ
基于Förster共振能量转移（FRET）的传感器几十年来一直是细胞生物学家工具包中的强大工具。通过对荧光蛋白，蛋白质-蛋白质相互作用和报告成分的结构生物学的基本理解，研究人员已经能够采用创造性的设计方法来构建传感器，这些传感器能够独特地探测活细胞中的各种现象，包括局部钙信号的可视化，亚细胞活性梯度和张力生成，仅举几例。虽然FRET传感器已显着影响了许多领域，人们还必须认识到的局限性，传统的，基于强度的FRET测量源于探针浓度的变化，对光漂白的敏感性，并通过FRET荧光团之间的渗出。荧光寿命成像显微镜（FLIM）在很大程度上克服了基于强度的FRET测量的局限性。一般而言，FLIM测量的是荧光团吸收光子和发射光子之间的时间，对于本章所述的报告分子，该时间为纳秒（ns）。当FLIM应用于FRET传感器（FLIM-FRET），测量供体荧光团的寿命提供了有价值的信息，如FRET效率和从事FRET报告的百分比。本章介绍了FLIM-FRET的基本原理，以告知该技术的实际应用，并使用两个已建立的FRET报告作为概念证明，概述了如何使用市售FLIM系统。
Förster resonance energy transfer (FRET)-based sensors have been powerful tools in cell biologists’ toolkit for decades. Informed by fundamental understanding of fluorescent proteins, protein-protein interactions, and the structural biology of reporter components, researchers have been able to employ creative design approaches to build sensors that are uniquely capable of probing a wide range of phenomena in living cells including visualization of localized calcium signaling, sub-cellular activity gradients, and tension generation to name but a few. While FRET sensors have significantly impacted many fields, one must also be cognizant of the limitations to conventional, intensity-based FRET measurements stemming from variation in probe concentration, sensitivity to photo-bleaching, and bleed-through between the FRET fluorophores. Fluorescence lifetime imaging microscopy (FLIM) largely overcomes the limitations of intensity based FRET measurements. In general terms, FLIM measures the time, which for the reporters described in this chapter is nanoseconds (ns), between photon absorption and emission by a fluorophore. When FLIM is applied to FRET sensors (FLIM-FRET), measurement of the donor fluorophore lifetime provides valuable information such as FRET efficiency and the percentage of reporters engaged in FRET. This chapter introduces fundamental principles of FLIM-FRET towards informing the practical application of the technique and, using two established FRET reporters as proofs of concept, outlines how to use a commercially available FLIM system.
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