FLIM Strategies for Intracellular Sensing Fluorescence Lifetime Imaging as a Tool to Quantify Analytes of Interest
FLIM Strategies for Intracellular Sensing Fluorescence Lifetime Imaging as a Tool to Quantify Analytes of Interest
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DOI：
10.1007/4243_2014_67
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Orte, Angel
中科院分区：
文献类型：
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作者：
Ruedas-Rama, Maria J.;Alvarez-Pez, Jose M.;Orte, Angel
从显微镜发展的最初几年起，科学家们就一直在追求观察活细胞活动的能力，以便在真实的时间内探测细胞内发生的过程。荧光显微镜已成为一种非凡的工具，用于解开与理解细胞生理学相关的无数细胞过程和相互作用。某些分析物的细胞内传感对于理解其中一些过程至关重要，例如细胞pH值和代谢状态之间的关系或特定离子在信号传导途径中的作用。然而，从细胞内部获取定量信息并不是一个微不足道的挑战。基于强度的比率荧光显微镜方法是常用的，但它们存在许多系统性困难，使其不适合定量传感。荧光寿命成像显微镜（FLIM）利用光子发射的多维性质检测荧光发射的持续时间。基于FLIM的细胞内传感方法，特别是在单光子定时（SPT）模式下的时域中，克服了荧光强度方法的许多局限性。在这里，我们审查策略FLIM为基础的细胞内传感的局部pH值，离子浓度和生物分子的相互作用。在第一部分中，我们展示了如何深入了解染料的物理性质可以在FLIM传感器的发展是有用的。然后，我们回顾了基于纳米颗粒的FLIM传感器的发展领域。最后，扩展检测能力的FRET和使用FLIM的大规模分析的组织进行了讨论。
Since the very early years of microscopy development, scientists have pursued the ability to observe live cellular activity in order to probe the processes occurring inside cells in real time. Fluorescence microscopy has become an extraordinary tool for unraveling the myriad cellular processes and interactions that are relevant to understanding cell physiology. The intracellular sensing of certain analytes is of crucial importance to understanding some of these processes, such as the relation between cellular pH and metabolic states or the roles of specific ions in signaling pathways. However, the acquisition of quantitative information from the interiors of cells is not a trivial challenge. Ratiometric, intensity-based fluorescence microscopy approaches are commonly used, but they suffer from many systematic difficulties that make them unsuitable for quantitative sensing. Fluorescence lifetime imaging microscopy (FLIM) detects the time duration of fluorescence emission, taking advantage of the multidimensional nature of photon emission. FLIM-based intracellular sensing approaches, especially in the time domain in single-photon timing (SPT) mode, overcome many of the limitations of fluorescence-intensity methods. Herein, we review strategies for the FLIM-based intracellular sensing of local pH, ion concentration, and biomolecular interactions. In the first section, we demonstrate how in-depth knowledge of the photophysics of dyes can be useful in the development of FLIM sensors. Then, we review the growing field of nanoparticle-based FLIM sensors. Finally, the expanding detection capabilities of FRET and the use of FLIM for the larger-scale analysis of tissues are discussed.