![]() A weaker endogenous source of fluorescence was also identified within mitochondria. This discovery has greatly advanced the study of energy distribution in biological systems. For example, mitochondria were identified using a small fluorescent molecule called tetramethyl-rhodamine-ethyl-ester (TMRE), which binds only to the mitochondrial membrane. Fluorescence-based targeting provided unique molecular specificity in high-resolution microscopy. By the late 20th century, numerous brightly fluorescent small molecules had been categorized and repurposed as markers bound to other molecules. 2 These experiments advanced fluorescence as a major means for biophysical and biochemical investigation. In the mid-20th century, Weber used fluorescence properties of molecules such as depolarization along with absorption and emission spectra to pinpoint molecular dynamics and reveal kinetic parameters for biologically relevant processes such as enzyme binding. 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. Stokes examined flower petals, leaves, turmeric, calcium fluoride, and many other compounds. 1 At that time, refraction (or refrangibility), internal dispersion, circular dichroism, and other phenomena of light were well studied. 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. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.įluorescence microscopy is a core biomedical imaging tool that provides high-resolution images of molecular contrast in living samples. Advantages and limitations of FLIM are also discussed.Ĭonclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. 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). Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.Īim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.Īpproach: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. 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. Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores.
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