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Related Concept Videos

Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

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Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...
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Variables Affecting Phosphorescence and Fluorescence01:26

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Fluorescence and phosphorescence are essential phenomena in fields like analytical chemistry, biological imaging, and materials science, where they detect molecular properties and visualize cellular structures. Understanding the variables that influence these luminescent behaviors is crucial for maximizing accuracy and efficiency in their applications. These variables can broadly be grouped into chemical structure, solvent properties, and external conditions, each playing a distinct role in...
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Total Internal Reflection Fluorescence Microscopy01:05

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Related Experiment Video

Updated: Jul 16, 2025

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
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Fluorescence molecular tomography for quantum yield and lifetime.

Wenxiang Cong, Ge Wang

    Applied Optics
    |September 14, 2023
    PubMed
    Summary

    This study introduces a new fluorescence molecular tomography (FMT) algorithm using photon-counting microcomputed tomography (PCMCT) for improved imaging of fluorescent markers in mice. The method enhances accuracy in estimating fluorescence lifetime and quantum yield, aiding in biological research and drug development.

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    Area of Science:

    • Biomedical Imaging
    • Optical Imaging
    • Medical Physics

    Background:

    • Fluorescence molecular tomography (FMT) is a key noninvasive imaging technique for internal fluorescence agents in biological tissues, particularly in small animal models.
    • Applications span diagnosis, therapy, and drug design, highlighting the need for improved resolution and accuracy in FMT.
    • Existing FMT methods face challenges with image reconstruction stability and accurately quantifying fluorescence parameters like quantum yield and lifetime.

    Purpose of the Study:

    • To develop and validate a novel fluorescent reconstruction algorithm for FMT.
    • To integrate time-resolved fluorescence imaging data with photon-counting microcomputed tomography (PCMCT) images.
    • To enhance the estimation accuracy of quantum yield and fluorescence lifetime of internal markers in a mouse model.

    Main Methods:

    • A new reconstruction algorithm was developed combining time-resolved fluorescence data with PCMCT images.
    • PCMCT images were utilized to provide prior knowledge of a permissible region of interest for fluorescence yield and lifetime.
    • This approach reduces unknown variables in the inverse problem, thereby improving image reconstruction stability.

    Main Results:

    • Numerical experiments demonstrated the accuracy and stability of the proposed reconstruction method, even with noisy data.
    • The algorithm achieved a reconstruction error of 0.02 ns for fluorescence lifetime.
    • An average relative error of 18% was obtained for quantum yield reconstruction.

    Conclusions:

    • The combined FMT and PCMCT approach significantly improves the stability and accuracy of reconstructing fluorescence parameters.
    • This method offers a more reliable tool for quantitative molecular imaging in preclinical research.
    • The enhanced accuracy in estimating quantum yield and lifetime has direct implications for drug development and disease diagnosis.