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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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Quantifying uncertainty in phasor-based time-domain fluorescence lifetime imaging microscopy.

Qinyi Chen1, Jongchan Park1, Shuqi Mu1

  • 1Department of Bioengineering, University of California, Los Angeles, California 90095, USA.

Biomedical Optics Express
|August 14, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a new model to understand noise in phasor-based time-domain fluorescence lifetime imaging microscopy (FLIM). The model quantifies how photon shot noise affects measurements, improving FLIM reliability in low-light conditions.

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Area of Science:

  • Biophotonics
  • Microscopy
  • Spectroscopy

Background:

  • Phasor analysis is a fit-free method for time-domain fluorescence lifetime imaging microscopy (FLIM).
  • Quantitative accuracy in FLIM is limited by noise, especially photon shot noise, affecting lifetime and unmixing.
  • Understanding noise propagation is crucial for reliable FLIM data.

Purpose of the Study:

  • To develop a theoretical uncertainty model for phasor-based time-domain FLIM.
  • To analytically capture the impact of photon shot noise on FLIM measurements.
  • To improve the quantitative accuracy and reliability of phasor-based FLIM.

Main Methods:

  • Developed a theoretical uncertainty model for phasor-based time-domain FLIM.
  • Analytically modeled the propagation of photon shot noise.
  • Validated the model using Monte Carlo simulations and experimental data from dyes and biological tissues.

Main Results:

  • The model accurately captures the propagation of shot noise in phasor coordinates.
  • Quantified the impact of noise on fluorophore weight estimation.
  • Demonstrated improved reliability in photon-limited imaging scenarios.

Conclusions:

  • The developed uncertainty model enhances the reliability of phasor-based time-domain FLIM.
  • This approach is particularly beneficial for photon-limited imaging applications.
  • The model provides a quantitative understanding of noise limitations in FLIM.