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

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.