SIGNIFICANCE Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. AIM We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. APPROACH This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. CONCLUSIONS FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

中文翻译：

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荧光寿命成像显微镜：仪器、分析和应用的基础知识和进展。

意义 荧光寿命成像显微镜 (FLIM) 是一种区分荧光团独特分子环境的强大技术。FLIM 测量荧光团在发射光子之前保持激发状态的时间，并检测单独使用光谱技术不明显的荧光团分子变化。FLIM 对多种生物医学过程敏感，包括疾病进展和药物疗效。目标 我们概述 FLIM 原理、仪器和分析，同时重点介绍最新发展和生物应用。方法 本综述涵盖了 FLIM 原理和理论，包括相对于基于强度的荧光测量的优点。总结了时域和频域 FLIM 仪器的基础知识以及最新发展。回顾了量化细胞异质性的空间和分子特征的图像分割和分析策略。最后，提供了代表性的应用，包括细胞和细胞器水平分子变化的高分辨率 FLIM、外源和内源荧光团的使用以及利用福斯特共振能量转移 (FRET) 成像蛋白质-蛋白质相互作用。还讨论了 FLIM 的优点和局限性。结论 FLIM 有利于探测荧光团的分子环境，以了解仅通过强度测量无法阐明的荧光团行为。FLIM 技术、分析和应用的开发将进一步推进生物学研究和临床评估。