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Fluorescence lifetime estimation: A practical approach using Flipper-TR FLIM.

Tithi Mandal1, Aurélien Roux1, Juan Manuel García-Arcos2

  • 1Department of Biochemistry, University of Geneva, Geneva, Switzerland.

Methods in Enzymology
|March 1, 2026
PubMed
Summary
This summary is machine-generated.

This study benchmarks methods for analyzing Flipper-TR membrane dye lifetime using fluorescence lifetime imaging microscopy (FLIM). It provides a framework for choosing the best lifetime estimation strategy for live-cell membrane tension studies.

Keywords:
Cell mechanicsFLIMFlipper-TRFluorescence lifetime imaging microscopyFrequency-domain lifetime analysisMembrane biophysicsMembrane tensionPhasor analysisTime-domain lifetime analysis

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Flipper-TR is a vital membrane dye for measuring live-cell membrane tension using fluorescence lifetime imaging microscopy (FLIM).
  • Optimal data analysis strategies for Flipper-TR lifetime extraction remain undefined, hindering quantitative FLIM applications.

Purpose of the Study:

  • To systematically compare and benchmark various methods for estimating Flipper-TR fluorescence lifetime.
  • To establish a decision-making framework for selecting optimal FLIM analysis strategies for Flipper dyes across diverse experimental conditions.

Main Methods:

  • Systematic comparison of Flipper-TR lifetime estimation techniques: multi-exponential reconvolution fitting, tail fitting, mean photon arrival time (first moment), and phasor analysis.
  • Evaluation of estimators under varying photon budgets, sample properties, microscope models, and laser frequencies.
  • Quantitative fluorescence lifetime imaging microscopy (FLIM) data acquisition and analysis.

Main Results:

  • Performance of different lifetime estimators varied significantly based on photon budget and experimental parameters.
  • Phasor analysis and mean photon arrival time showed robustness across tested conditions.
  • No single method universally outperformed others, highlighting the need for context-specific selection.

Conclusions:

  • A comprehensive benchmark for Flipper-TR lifetime analysis in FLIM is provided.
  • Guidance is offered for selecting appropriate quantitative FLIM analysis strategies based on experimental context.
  • Standardized analysis protocols are crucial for reproducible Flipper dye-based membrane tension measurements.