Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
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DOI：
10.1117/1.jbo.25.7.071203
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发表时间：
2020-07-01
影响因子：
3.5
通讯作者：
Skala, Melissa C.
中科院分区：
文献类型：
--
作者：
Datta, Rupsa;Heaster, Tiffany M.;Skala, Melissa C.
意义：荧光寿命成像显微镜（FLIM）是一种强有力的技术，以区分独特的分子环境的荧光团。FLIM测量荧光团在发射光子之前保持在激发态的时间，并检测单独使用光谱技术不明显的荧光团的分子变化。FLIM是敏感的多个生物医学过程，包括疾病的进展和药物efficacy.Aim：我们提供了一个概述FLIM的原则，仪器和分析，同时突出了最新的发展和生物学applications.Approach：这篇评论涵盖FLIM的原则和理论，包括基于强度的荧光测量的优势。FLIM仪器在时域和频域的基本原理进行了总结，沿着最近的发展。图像分割和分析策略，量化的空间和分子特征的细胞异质性进行审查。最后，提供了代表性的应用，包括高分辨率FLIM的细胞和细胞器水平的分子变化，使用外源性和内源性荧光团，和成像蛋白质-蛋白质相互作用与福斯特共振能量转移（FRET）。FLIM的优点和局限性也discussed.Conclusions：FLIM是有利的探测荧光团的分子环境，以告知荧光团的行为，不能单独用强度测量来阐明。FLIM技术、分析和应用的发展将进一步推动生物学研究和临床评估。(C)作者。由SPIE在知识共享署名4.0未移植许可下发布。
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 Forster 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. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.