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Fluorescence and Phosphorescence: Instrumentation01:25

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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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Quantifying Uncertainty in Phasor-Based Time-Domain Fluorescence Lifetime Imaging Microscopy.

Qinyi Chen, Jongchan Park, Shuqi Mu

    Biorxiv : the Preprint Server for Biology
    |April 28, 2025
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    Summary
    This summary is machine-generated.

    This study introduces a new uncertainty model for phasor-based time-domain fluorescence lifetime imaging microscopy (FLIM). It quantifies how photon shot noise impacts FLIM analysis, improving reliability in low-light conditions.

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    Area of Science:

    • Biophotonics
    • Microscopy
    • Spectroscopy

    Background:

    • Phasor analysis offers a fit-free method for time-domain fluorescence lifetime imaging microscopy (FLIM).
    • Noise, especially photon shot noise, limits the quantitative accuracy of phasor-based FLIM.
    • Shot noise introduces variability and bias in lifetime estimation and fluorophore unmixing.

    Purpose of the Study:

    • To develop a theoretical uncertainty model for phasor-based time-domain FLIM.
    • To analytically capture the propagation of shot noise in FLIM data.
    • To quantify the impact of shot noise on phasor coordinates and fluorophore weight estimation.

    Main Methods:

    • Development of a theoretical uncertainty model for phasor-based time-domain FLIM.
    • Analytical derivation of shot noise propagation.
    • Validation using Monte Carlo simulations.
    • Experimental validation with fluorescent dyes and biological tissues.

    Main Results:

    • The developed model analytically quantifies shot noise propagation in phasor FLIM.
    • The model accurately predicts the impact of noise on phasor coordinates.
    • Estimation of fluorophore weights is shown to be affected by shot noise.
    • Model validation confirmed its accuracy with simulated and experimental data.

    Conclusions:

    • The theoretical uncertainty model enhances the reliability of phasor-based time-domain FLIM.
    • The model improves the efficiency of FLIM analysis, especially in photon-limited scenarios.
    • This work provides a quantitative framework for understanding and mitigating noise in FLIM.