Abstract
A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein-protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.
Highlights
In the past decade, there has been a surge of interest in imaging methodologies capable of measuring luminescence lifetime, which previously had been restricted to a small community of physicists and engineers
fluorescence lifetime imaging (FLI) methodologies can be integrated into traditional fluorescence imaging approaches, including widefield, confocal, multiphoton, super-resolution and light-sheet microscopy, as well as macroimaging, fluorescence molecular tomography (FMT) and other optical imaging-based approaches (Ishikawa-Ankerhold et al, 2012; Meyer-Almes, 2017; Sarder et al, 2015; Denicke et al, 2007; Becker et al, 2017; Hirvonen and Suhling, 2020; Le Marois and Suhling, 2017; Datta et al, 2020; Poudel et al, 2020; Wang et al, 1992)
Modern FLI and phosphorescence lifetime microscopy (PLIM) platforms cover an optical resolution ranging from sub-micrometer to several millimeters, a spectral range from 180 to 1700 nm and a time-domain resolution from sub-picoseconds to milliseconds, with measurement speeds that range from real-time for one-photon FLI and PLIM (Raspe et al, 2016; Trinh et al, 2019; Cheng et al, 2020) to several minutes per frame for two-photon PLIM (Rytelewski et al, 2019)
Summary
There has been a surge of interest in imaging methodologies capable of measuring luminescence lifetime, which previously had been restricted to a small community of physicists and engineers. Depending on its molecular structure, a luminophore can exhibit either fluorescence (with a lifetime of pico- to nano-seconds) or phosphorescence (with a lifetime of micro- to milli-seconds), and the methods to observe these two are designated as fluorescence lifetime imaging (FLI) and phosphorescence lifetime microscopy (PLIM), respectively (Fig. 1A). FLI methodologies can be integrated into traditional fluorescence imaging approaches, including widefield, confocal, multiphoton, super-resolution and light-sheet microscopy, as well as macroimaging, fluorescence molecular tomography (FMT) and other optical imaging-based approaches (Ishikawa-Ankerhold et al, 2012; Meyer-Almes, 2017; Sarder et al, 2015; Denicke et al, 2007; Becker et al, 2017; Hirvonen and Suhling, 2020; Le Marois and Suhling, 2017; Datta et al, 2020; Poudel et al, 2020; Wang et al, 1992). Multiparameter and often non-invasive and nondestructive three-dimensional (3D) analyses of live cells and tissues to be performed, FLI and PLIM provide a broad support to the ongoing ‘3D molecular-imaging revolution’
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