Probing biological processes in living organisms that could provide one-of-a-kind insights into real-time alterations of significant physiological parameters is a formidable task that calls for specialized analytic devices. Classical biochemical methods have significantly aided our understanding of the mechanisms that regulate essential biological processes. These methods, however, are typically insufficient for investigating transient molecular events since they focus primarily on the end outcome. Fluorescence resonance energy transfer (FRET) microscopy is a potent tool used for exploring non-invasively real-time dynamic interactions between proteins and a variety of biochemical signaling events using sensors that have been meticulously constructed. Due to their versatility, FRET-based sensors have enabled the rapid and standardized assessment of a large array of biological variables, facilitating both high-throughput research and precise subcellular measurements with exceptional temporal and spatial resolution. This review commences with a brief introduction to FRET theory and a discussion of the fluorescent molecules that can serve as tags in different sensing modalities for studies in chemical biology, followed by an outlining of the imaging techniques currently utilized to quantify FRET highlighting their strengths and shortcomings. The article also discusses the various donor-acceptor combinations that can be utilized to construct FRET scaffolds. Specifically, the review provides insights into the latest real-time bioimaging applications of FRET-based sensors and discusses the common architectures of such devices. There has also been discussion of FRET systems with multiplexing capabilities and multi-step FRET protocols for use in dual/multi-analyte detections. Future research directions in this exciting field are also mentioned, along with the obstacles and opportunities that lie ahead.

中文翻译：

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探索基于 FRET 的分子传感器的前景：新兴应用的设计策略和最新进展


探测生物体中的生物过程，为重要生理参数的实时变化提供独一无二的见解，是一项艰巨的任务，需要专门的分析设备。经典的生化方法极大地帮助我们理解调节基本生物过程的机制。然而，这些方法通常不足以研究瞬时分子事件，因为它们主要关注最终结果。荧光共振能量转移 （FRET） 显微镜是一种有效的工具，用于使用精心构建的传感器来探索蛋白质与各种生化信号事件之间的非侵入性实时动态相互作用。由于其多功能性，基于 FRET 的传感器能够快速、标准化地评估大量生物变量，从而以卓越的时间和空间分辨率促进高通量研究和精确的亚细胞测量。本综述首先简要介绍了 FRET 理论，并讨论了可以在化学生物学研究的不同传感模式中用作标签的荧光分子，然后概述了目前用于量化 FRET 的成像技术，强调了它们的优点和缺点。本文还讨论了可用于构建 FRET 支架的各种供体-受体组合。具体来说，该综述提供了对基于 FRET 的传感器的最新实时生物成像应用的见解，并讨论了此类设备的常见架构。还讨论了具有多重检测功能的 FRET 系统和用于双重/多分析物检测的多步骤 FRET 方案。 还提到了这个令人兴奋的领域的未来研究方向，以及未来的障碍和机遇。