Abstract
.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.
Highlights
Fluorescence microscopy is a core biomedical imaging tool that provides high-resolution images of molecular contrast in living samples
Fluorescence lifetime imaging microscopy (FLIM) is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone
We review FLIM instrumentation and FLIM analysis methods including segmentation and population density modeling of cell heterogeneity, and we close with a summary of FLIM applications in vivo and in vitro
Summary
Fluorescence microscopy is a core biomedical imaging tool that provides high-resolution images of molecular contrast in living samples. Stokes coined the term “fluorescence” in 1852 for the “remarkable phenomena of light” observed in the materials that emitted light at a different color than the absorbed light.. Refraction (or refrangibility), internal dispersion, circular dichroism, and other phenomena of light were well studied. Leaves, turmeric, calcium fluoride, and many other compounds. In the 20th century, fluorescence was redefined as a short-lived emission of photons caused by the incidence of higher energy photons and became a popular tool for studying molecular dynamics and characterizing compounds
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